Micrornas in neurodegenerative disorders

ABSTRACT

Provided herein are methods of treating a neurodegenerative disorder such as Amyotrophic Lateral Sclerosis (ALS) or Multiple Sclerosis (MS) that include administering to a subject at least one inhibitory nucleic acid that decreases the level or activity of microRNA hsa-miR-155.

Cross-Reference to Related Applications

This application is a continuation of U.S. patent application Ser. No.14/350,977, filed Apr. 10, 2014, which is a U.S. National PhaseApplication under 35 U.S.C. § 371 of International Patent ApplicationNo. PCT/US2012/059671, filed Oct. 11, 2012, which claims priority toU.S. Provisional Patent Application No. 61/601,205, filed Feb. 21, 2012,and U.S. Provisional Patent Application No. 61/545,968, filed Oct. 11,2011, each of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Inflammation has been implicated in a number of neurodegenerativedisorders (e.g., amyotrophic lateral sclerosis (ALS) and multiplesclerosis). For example, increased inflammatory responses have beenobserved in both human ALS patients and animal models of ALS (McGreer etal., Muscle Nerve 26:459-470, 2002; Beers et al., Proc. Natl. Acad. Sci.U.S.A. 105:15558-15563, 2008; Banerjee et al., PLoS ONE 3:e2740, 2008;Chiu et al., Proc. Natl. Acad. Sci. U.S.A. 105:17913-17918, 2008; Chiuet al., Proc. Natl. Acad. Sci. U.S.A. 106:20960-20965, 2009; Beers etal., Proc. Natl. Acad. Sci. U.S.A. 103:16021-16026, 2006; Henkel et al.,Ann. Neurol. 55:221-235, 2004; Meissner et al., Proc. Natl. Acad. Sci.U.S.A. 107:13046-13050, 2010). It has been reported that both microgliaand astrocytes are activated in the central nervous system in a mousemodel of familial ALS (Alexianu et al., Neurology 57:1282-1289, 2001;Hall et al., Glia 23:249-256, 1998), and that natural killer cells andperipheral T-cells infiltrate the spinal cord during neurodegenerativedisease progression in a mouse model of ALS (Chiu et al., Proc. Natl.Acad. Sci. U.S.A. 105:17913-17918, 2008).

In the peripheral nervous system, degeneration of peripheral motor axonsis an early and significant pathological feature in ALS patients and inanimal models of ALS, and is preceded by the recruitment and activationof macrophages (Chiu et al., Proc. Natl. Acad. Sci. U.S.A.106:20960-20965, 2009). A specific monocyte subset (Ly6C^(Hi)) in miceparticipates in tissue damage and disease pathogenesis in a mouse modelsof multiple sclerosis (King et al., Blood 113:3190-3197, 2009), andthese monocytes are recruited to inflamed tissues by CCL2 (Kim et al.,Immunity 34:769-780, 2011; Getts et al., J. Exp. Med. 205:2319-2337,2008).

SUMMARY OF THE INVENTION

The invention is based, at least in part, on the discovery that specificmicroRNAs and inflammatory marker genes are increased or decreased inthe cerebrospinal fluid (CSF) and in CD14⁺CD16⁻and CD14⁺CD16⁺ monocytesfrom subjects having neurodegenerative diseases compared to theexpression level of these microRNAs and these inflammatory marker genesin the CSF and in CD14⁺CD16⁻ and CD14⁺CD16⁺ monocytes from healthysubjects. The specific microRNAs and inflammatory marker genes that havebeen identified as being increased or decreased in the CSF and/or inCD14⁺CD16⁻ and/or CD14⁺CD16⁺ monocytes in subjects having aneurodegenerative disease are listed in Tables 1-21. The inflammatorymarkers as described herein are listed in Tables 20 and 21.

Provided herein are methods of diagnosing a neurodegenerative disorder(e.g., amyotrophic lateral sclerosis or multiple sclerosis) in a subjectthat include determining a level of one or more microRNAs and/or one ormore inflammatory markers listed in any one or more of Tables 1-21 in amonocyte (e.g., CD14⁺CD16⁻ and CD14⁺CD16⁺ monocyte) or in CSF from thesubject, and comparing the level of the one or more microRNAs and/or oneor more inflammatory markers to a reference level of the one or moremicroRNAs and/or one or more inflammatory markers (e.g., a thresholdlevel or a level present in the CSF, or a CD14⁺CD16⁻ or a CD14⁺CD16⁺monocyte from a healthy subject). In these methods, an increase ordecrease in the level of the one or more microRNAs and/or the one ormore inflammatory markers relative to the reference level indicates thatthe subject has a neurodegenerative disorder.

Also provided are methods of identifying a subject at risk of developinga neurodegenerative disorder (e.g., amyotrophic lateral sclerosis ormultiple sclerosis) that include determining a level of one or moremicroRNAs and/or one or more inflammatory markers listed in any one ormore of Tables 1-21 in a monocyte (e.g., CD14⁺CD16⁻ or CD14⁺CD16⁺monocyte (e.g., a peripheral or blood-derived monocyte) or CSF from thesubject, and comparing the level of the one or more microRNAs and/or theone or more inflammatory markers to a reference level of the one or moremicroRNAs and/or the one or more inflammatory markers (e.g., a thresholdlevel or a level present in the CSF, or a CD14⁺CD16⁻ or CD14⁺CD16⁺monocyte (e.g., a peripheral or blood-derived monocyte) from a healthysubject). In these methods, an increase or decrease in the level of theone or more microRNAs and/or the one or more inflammatory markersrelative to the reference level indicates that the subject has anincreased or decreased risk of developing a neurodegenerative disorder(e.g., relative to a person who does not show an increase or decrease inthe level of the one or more microRNAs and/or the one or moreinflammatory markers relative to a reference level).

Also provided are methods of predicting the rate of disease progressionin a subject having a neurodegenerative disorder (e.g., amyotrophiclateral sclerosis or multiple sclerosis) that include determining alevel of one or more microRNAs and/or one or more inflammatory markerslisted in any one or more of Tables 1-21 in a monocyte (e.g., CD14⁺CD16⁻or CD14⁺CD16⁺ monocyte (e.g., a peripheral or blood-derived monocyte) orCSF from the subject, and comparing the level of the one or moremicroRNAs and/or the one or more inflammatory markers to a referencelevel of the one or more microRNAs and/or the one or more inflammatorymarkers (e.g., a threshold level or a level present in the CSF or aCD14⁺CD16⁻ or CD14⁺CD16⁻ monocyte (e.g., a peripheral or blood-derivedmonocyte) from a healthy subject). In these methods, an increase ordecrease in the level of the one or more microRNAs and/or the one ormore inflammatory markers relative to the reference level indicates thatthe subject will have an increased or decreased rate of diseaseprogression (e.g., relative to a person who does not show an increase ordecrease in the level of the one or more microRNAs and/or the one ormore inflammatory markers relative to a reference level).

Also provided are methods of selecting a subject for treatment of aneurodegenerative disorder (e.g., amyotrophic lateral sclerosis ormultiple sclerosis) that include determining a level of one or moremicroRNAs and/or one or more inflammatory markers listed in any one ormore of Tables 1-21 in a monocyte (e.g., a CD14⁺CD16⁻ or CD14⁺CD16⁺monocyte (e.g., a periperhal or blood-derived monocyte) or CSF from thesubject; comparing the level of the one or more microRNAs and/or the oneor more inflammatory markers to a reference level of the one or moremicroRNAs and/or the one or more inflammatory markers (e.g., a thresholdlevel or a level present in the CSF, or a CD14⁺CD16⁻ or CD14⁺CD16⁺monocyte (e.g., a peripheral or blood-derived monocyte) from a healthysubject); and selecting a subject having an increase or decrease in thelevel of the one or more microRNAs and/or the one or more inflammatorymarkers relative to the reference level for treatment of aneurodegenerative disorder.

Also provided are methods of determining the efficacy of treatment of aneurodegenerative disorder (e.g., amyotrophic lateral sclerosis ormultiple sclerosis) in a subject that include determining a level of oneor more microRNAs and/or one or more inflammatory markers listed in anyone or more of Tables 1-21 in a monocyte (e.g., a CD14⁺CD16⁻ orCD14⁺CD16⁺ monocyte (e.g., a peripheral or blood-derived monocyte)) orCSF from the subject at a first time point; determining a level of theone or more microRNAs and/or the one or more inflammatory markers in amonocyte (e.g., a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte (e.g., a peripheralor blood-derived monocyte) or CSF from the subject at a second timepoint following administration of at least one dose of a treatment; andcomparing the level of the one or more microRNAs and/or the one or moreinflammatory markers at the first time point to the level of the one ormore microRNAs and/or the one or more inflammatory markers at the secondtime point. In these methods, a return or approach to levels in ahealthy subject at the second time point (e.g., a decrease or increasein the level of the one or more microRNAs and/or the one or moreinflammatory markers at the second time point compared to the levels atthe first time point as described herein) indicates that the treatmentwas effective in the subject (e.g., the treatment was effective relativeto a subject having the same neurodegenerative disorder and receivingthe same treatment, but does not show a return or approach to levels ina healthy subject at the second time point (e.g., an increase ordecrease in the level of the one or more microRNAs and/or the one ormore inflammatory markers compared to a reference value as describedherein), or does not show as significant of an increase or decrease inthe level of the one or more microRNAs and/or the one or moreinflammatory markers compared to a reference value as described herein).

Also provided are methods for selecting a subject for participation in aclinical study. These methods include determining a level of one or moremicroRNAs and/or one or more inflammatory markers listed in any one ormore of Tables 1-21 in a monocyte (e.g., CD14⁺CD16⁻ or CD14⁺CD16⁺monocyte (e.g., a peripheral or blood-derived monocyte) or CSF from thesubject; comparing the level of the one or more microRNAs and/or the oneor more inflammatory markers to a reference level of the one or moremicroRNAs and/or the one or more inflammatory markers (e.g., a thresholdlevel or a level present in the CSF, or a CD14⁺CD16⁻ or CD14⁺CD16⁺monocyte (e.g., a peripheral or blood-derived monocyte) from a healthysubject); and selecting a subject having an increase or a decrease inthe level of the one or more microRNAs and/or the one or more of theinflammatory markers compared to the reference level for participationin a clinical study.

Also provided are methods of treating a neurodegenerative disorder(e.g., amyotrophic lateral sclerosis or multiple sclerosis) in a subjectthat include administering to a subject at least one agent (e.g., aninhibitory nucleic acid, e.g., an antagomir) that decreases theexpression or activity of one or more of the microRNAs listed in Tables1, 3, 5, 7, 9, 11, 12, 14, 16, or 18, or any of the inflammatory markerslisted in Table 21. Also provided are methods of treating aneurodegenerative disorder (e.g., amyotrophic lateral sclerosis ormultiple sclerosis) in a subject that include administering to a subjectat least one agent (e.g., a nucleic acid containing a sense(protein-encoding) nucleic acid) that increases the expression oractivity of one or more of the microRNAs listed in Tables 2, 4, 6, 8,10, 13, 15, 17, or 19, and/or one or more of the inflammatory markerslisted in Table 20.

Also provided are methods of treating a neurodegenerative disorder(e.g., ALS, such as sporadic ALS or familial ALS, or multiple sclerosis)in a subject that include administering to a subject having aneurodegenerative disorder (e.g., ALS, such as sporadic ALS or familialALS, or multiple sclerosis) at least one inhibitory nucleic acid (e.g.,siRNA, an antisense oligonucleotide, an antagomir, and/or a ribozyme)comprising a sequence that is complementary to a contiguous sequencepresent in hsa-miR-155 (e.g., a contiguous sequence present in theprecursor or mature form of hsa-miR-155).

Also provided is an inhibitory nucleic acid comprising a sequence thatis complementary to a contiguous sequence, e.g., a contiguous sequenceof at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, or 25 nucleotides, present in hsa-miR-155, hsa-miR-19b,hsa-miR-106b, hsa-miR-30b, hsa-miR-21, hsa-miR-142-5p, hsa-miR-27a,hsa-miR-16, hsa-miR-374a, hsa-miR-374b, hsa-miR-101, hsa-miR-340,hsa-miR-30e, hsa-miR-29c, hsa-miR-29a, hsa-miR-223, hsa-miR-26a,hsa-miR-26b, hsa-miR-24, hsa-miR-181a, hsa-miR-103, hsa-miR-532-3p,hsa-miR-27b, hsa-miR-99b, hsa-miR-146a, hsa-miR-150, hsa-miR-328,hsa-miR-15b, or hsa-miR-19a, for use in treating amyotrophic lateralsclerosis (ALS) in a subject. Preferably the sequence is complementaryto a contiguous sequence of at least 7 or 8 nucleotides present inhsa-miR-155.

Provided herein are methods of diagnosing amyotrophic lateral sclerosis(ALS) in a subject that include: determining a level of one or moremicroRNAs selected from the group consisting of: hsa-miR-19b,hsa-miR-106b, hsa-miR-30b, hsa-miR-21, hsa-miR-142-5p, hsa-miR-27a,hsa-miR-16, hsa-miR-374a, hsa-miR-374b, hsa-miR-101, hsa-miR-340,hsa-miR-30e, hsa-miR-29c, hsa-miR-29a, hsa-miR-223, hsa-miR-26a,hsa-miR-26b, hsa-miR-24, hsa-miR-181a, hsa-miR-103, hsa-miR-155,hsa-miR-532-3p, hsa-miR-518f, hsa-miR-206, hsa-miR-204, hsa-miR-137,hsa-miR-453, hsa-miR-146a, hsa-miR-603, hsa-miR-1297, hsa-miR-192,hsa-miR-526a, hsa-miR-615-5p, hsa-miR-655, hsa-miR-450b-5p,hsa-miR-548b-3p, hsa-miR-584, hsa-miR-548f, hsa-miR-300, hsa-miR-302c,hsa-miR-328, hsa-miR-421, and hsa-miR-580 in a CD14⁺CD16⁻ monocyte fromthe subject; and comparing the level of the one or more of microRNA(s)in a CD14⁺CD16⁻ monocyte from the subject with a reference level of theone or more microRNA(s); whereby an increase in the level of one or moreof hsa-miR-19b, hsa-miR-106b, hsa-miR-30b, hsa-miR-21, hsa-miR-142-5p,hsa-miR-27a, hsa-miR-16, hsa-miR-374a, hsa-miR-374b, hsa-miR-101,hsa-miR-340, hsa-miR-30e, hsa-miR-29c, hsa-miR-29a, hsa-miR-223,hsa-miR-26a, hsa-miR-26b, hsa-miR-24, hsa-miR-181a, hsa-miR-103,hsa-miR-155, and hsa-miR-532-3p and/or a decrease in the level of one ormore of hsa-miR-518f, hsa-miR-206, hsa-miR-204, hsa-miR-137,hsa-miR-453, hsa-miR-146a, hsa-miR-603, hsa-miR-1297, hsa-miR-192,hsa-miR-526a, hsa-miR-615-5p, hsa-miR-655, hsa-miR-450b-5p,hsa-miR-548b-3p, hsa-miR-584, hsa-miR-548f, hsa-miR-300, hsa-miR-302c,hsa-miR-328, hsa-miR-421, and hsa-miR-580 in a CD14⁺CD16⁻ monocyte fromthe subject as compared to the reference level indicates that thesubject has ALS.

Also provided are methods of diagnosing amyotrophic lateral sclerosis(ALS) in a subject that include: determining a level of one or more ofhsa-miR-27b, hsa-miR-99b, hsa-miR-146a, hsa-miR-150, hsa-miR-328, andhsa-miR-532-3p in cerebrospinal fluid (CSF) in a subject; and comparingthe level of one or more of hsa-miR-27b, hsa-miR-99b, hsa-miR-146a,hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p in the CSF of the subjectto a reference level of the one or more of hsa-miR-27b, hsa-miR-99b,hsa-miR-146a, hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p, whereby anincrease in the level of one or more of hsa-miR-27b, hsa-miR-99b,hsa-miR-146a, hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p in the CSF ofthe subject compared to the reference level indicates that the subjecthas ALS.

Also provided are methods of diagnosing familial amyotrophic lateralsclerosis (ALS) in a subject that include: determining a level ofhsa-miR-27b and a level of one or more of hsa-miR-99b, hsa-miR-146a,hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p in cerebrospinal fluid(CSF) of a subject; and comparing the level of hsa-miR-27b in the CSF ofthe subject to a reference level of hsa-miR-27b, and the level of one ormore of hsa-miR-99b, hsa-miR-146a, hsa-miR-150, hsa-miR-328, andhsa-miR-532-3p in the CSF of the subject to a reference level of one ormore of hsa-miR-99b, hsa-miR-146a, hsa-miR-150, hsa-miR-328, andhsa-miR-532-3p; whereby an increase in the level of hsa-miR-27b in theCSF of the subject compared to the reference level of hsa-miR-27b, and adecrease or no significant change in the level of one or more ofhsa-miR-99b, hsa-miR-146a, hsa-miR-150, hsa-miR-328, and hsa-miR-532-3pin the CSF of the subject compared to the reference level of one or moreof hsa-miR-99b, hsa-miR-146a, hsa-miR-150, hsa-miR-328, andhsa-miR-532-3p indicates that the subject has familial ALS.

Also provided are methods of diagnosing sporadic amyotrophic lateralsclerosis (ALS) in a subject that include: determining a level of two ormore microRNAs selected from the group consisting of hsa-miR-27b,hsa-miR-99b, hsa-miR-146a, hsa-miR-150, hsa-miR-328, and hsa-miR-532-3pin cerebrospinal fluid (CSF) of a subject; and comparing the level ofthe two or more microRNAs in the CSF of the subject to a reference levelof the two or more microRNAs; whereby an increase in the level of thetwo or more microRNAs in the CSF of the subject compared to thereference level indicates that the subject has sporadic ALS.

Also provided are methods of identifying a subject at risk of developingamyotrophic lateral sclerosis (ALS) that include: determining a level ofone or more microRNAs selected from the group consisting of:hsa-miR-19b, hsa-miR-106b, hsa-miR-30b, hsa-miR-21, hsa-miR-142-5p,hsa-miR-27a, hsa-miR-16, hsa-miR-374a, hsa-miR-374b, hsa-miR-101,hsa-miR-340, hsa-miR-30e, hsa-miR-29c, hsa-miR-29a, hsa-miR-223,hsa-miR-26a, hsa-miR-26b, hsa-miR-24, hsa-miR-181a, hsa-miR-103,hsa-miR-155, hsa-miR-532-3p, hsa-miR-518f, hsa-miR-206, hsa-miR-204,hsa-miR-137, hsa-miR-453, hsa-miR-146a, hsa-miR-603, hsa-miR-1297,hsa-miR-192, hsa-miR-526a, hsa-miR-615-5p, hsa-miR-655, hsa-miR-450b-5p,hsa-miR-548b-3p, hsa-miR-584, hsa-miR-548f, hsa-miR-300, hsa-miR-302c,hsa-miR-328, hsa-miR-421, and hsa-miR-580 in a CD14⁺CD16⁻ monocyte fromthe subject; and comparing the level of the one or more microRNAs in aCD14⁺CD16⁻ monocyte from the subject with a reference level of the oneor more microRNAs; whereby an increase in the level of one or more ofhsa-miR-19b, hsa-miR-106b, hsa-miR-30b, hsa-miR-21, hsa-miR-142-5p,hsa-miR-27a, hsa-miR-16, hsa-miR-374a, hsa-miR-374b, hsa-miR-101,hsa-miR-340, hsa-miR-30e, hsa-miR-29c, hsa-miR-29a, hsa-miR-223,hsa-miR-26a, hsa-miR-26b, hsa-miR-24, hsa-miR-181a, hsa-miR-103,hsa-miR-155, and hsa-miR-532-3p and/or a decrease in the level of one ormore of hsa-miR-518f, hsa-miR-206, hsa-miR-204, hsa-miR-137,hsa-miR-453, hsa-miR-146a, hsa-miR-603, hsa-miR-1297, hsa-miR-192,hsa-miR-526a, hsa-miR-615-5p, hsa-miR-655, hsa-miR-450b-5p,hsa-miR-548b-3p, hsa-miR-584, hsa-miR-548f, hsa-miR-300, hsa-miR-302c,hsa-miR-328, hsa-miR-421, and hsa-miR-580 in a CD14⁺CD16⁻ monocyte fromthe subject as compared to the reference level indicates that thesubject has an increased risk of developing ALS.

Also provided are methods of identifying a subject at risk of developingamyotrophic lateral sclerosis (ALS) in a subject that include:determining a level of one or more of hsa-miR-27b, hsa-miR-99b,hsa-miR-146a, hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p incerebrospinal fluid (CSF) in a subject; and comparing the level of oneor more of hsa-miR-27b, hsa-miR-99b, hsa-miR-146a, hsa-miR-150,hsa-miR-328, and hsa-miR-532-3p in the CSF of the subject to a referencelevel of the one or more of hsa-miR-27b, hsa-miR-99b, hsa-miR-146a,hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p, whereby an increase in thelevel of one or more of hsa-miR-27b, hsa-miR-99b, hsa-miR-146a,hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p in the CSF of the subjectcompared to the reference level indicates that the subject has anincreased risk of developing ALS.

Also provided are methods of identifying a subject at risk of developingfamilial amyotrophic lateral sclerosis (ALS) that include: determining alevel of hsa-miR-27b and a level of one or more of hsa-miR-99b,hsa-miR-146a, hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p incerebrospinal fluid (CSF) of a subject; and comparing the level ofhsa-miR-27b in the CSF of the subject to a reference level ofhsa-miR-27b, and the level of one or more of hsa-miR-99b, hsa-miR-146a,hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p in the CSF of the subjectto a reference level of one or more of hsa-miR-99b, hsa-miR-146a,hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p, whereby an increase in thelevel of hsa-miR-27b in the CSF of the subject compared to the referencelevel of hsa-miR-27b, and a decrease or no significant change in thelevel of one or more of hsa-miR-99b, hsa-miR-146a, hsa-miR-150,hsa-miR-328, and hsa-miR- 532-3p in the CSF of the subject compared tothe reference level of one or more of hsa-miR-99b, hsa-miR-146a,hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p indicates that the subjecthas an increased risk of developing familial ALS.

Also provided are methods of identifying a subject at risk of developingsporadic amyotrophic lateral sclerosis (ALS) that include: determining alevel of two or more microRNAs selected from the group consisting ofhsa-miR-27b, hsa-miR-99b, hsa-miR-146a, hsa-miR-150, hsa-miR-328, andhsa-miR-532-3p in cerebrospinal fluid (CSF) of a subject; and comparingthe level of the two or more microRNAs in the CSF of the subject with areference level of the two or more microRNAs, whereby an increase in thelevel of the two or more microRNAs in the CSF of the subject compared tothe reference level indicates that the subject has an increased risk ofdeveloping sporadic ALS.

Also provided are methods of predicting the rate of disease progressionin a subject having amyotrophic lateral sclerosis (ALS) that include:determining a level of one or more microRNAs selected from the groupconsisting of: hsa-miR-19b, hsa-miR-106b, hsa-miR-30b, hsa-miR-21,hsa-miR-142-5p, hsa-miR-27a, hsa-miR-16, hsa-miR-374a, hsa-miR-374b,hsa-miR-101, hsa-miR-340, hsa-miR-30e, hsa-miR-29c, hsa-miR-29a,hsa-miR-223, hsa-miR-26a, hsa-miR-26b, hsa-miR-24, hsa-miR-181a,hsa-miR-103, hsa-miR-155, hsa-miR-532-3p, hsa-miR-518f, hsa-miR-206,hsa-miR-204, hsa-miR-137, hsa-miR-453, hsa-miR-146a, hsa-miR-603,hsa-miR-1297, hsa-miR-192, hsa-miR-526a, hsa-miR-615-5p, hsa-miR-655,hsa-miR-450b-5p, hsa-miR-548b-3p, hsa-miR-584, hsa-miR-548f,hsa-miR-300, hsa-miR-302c, hsa-miR-328, hsa-miR-421, hsa-miR-580,hsa-miR-15b, and hsa-miR19a in a CD14⁺CD16⁻ monocyte from the subject;and comparing the level of the one or more microRNAs in a CD14⁺CD16⁻monocyte from the subject to a reference level of the one or moremicroRNAs; whereby an increase in the level of one or more ofhsa-miR-19b, hsa-miR-106b, hsa-miR-30b, hsa-miR-21, hsa-miR-142-5p,hsa-miR-27a, hsa-miR-16, hsa-miR-374a, hsa-miR-374b, hsa-miR-101,hsa-miR-340, hsa-miR-30e, hsa-miR-29c, hsa-miR-29a, hsa-miR-223,hsa-miR-26a, hsa-miR-26b, hsa-miR-24, hsa-miR-181a, hsa-miR-103,hsa-miR-155, hsa-miR-532-3p, hsa-miR-15b, and miR-19a and/or a decreasein the level of one or more of hsa-miR-518f, hsa-miR-206, hsa-miR-204,hsa-miR-137, hsa-miR-453, hsa-miR-146a, hsa-miR-603, hsa-miR-1297,hsa-miR-192, hsa-miR-526a, hsa-miR-615-5p, hsa-miR-655, hsa-miR-450b-5p,hsa-miR-548b-3p, hsa-miR-584, hsa-miR-548f, hsa-miR-300, hsa-miR-302c,hsa-miR-328, hsa-miR-421, and hsa-miR-580 in a CD14⁺CD16⁻ monocyte fromthe subject as compared to the reference level indicates that thesubject will have an increased rate of disease progression.

Also provided are methods of predicting the rate of disease progressionin a subject having amyotrophic lateral sclerosis (ALS) that include:determining a level of one or more of hsa-miR-27b, hsa-miR-99b,hsa-miR-146a, hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p incerebrospinal fluid (CSF) in a subject; and comparing the level of oneor more of hsa-miR-27b, hsa-miR-99b, hsa-miR-146a, hsa-miR-150,hsa-miR-328, and hsa-miR-532-3p in the CSF of the subject to a referencelevel of the one or more of hsa-miR-27b, hsa-miR-99b, hsa-miR-146a,hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p, whereby an increase in thelevel of one or more of hsa-miR-27b, hsa-miR-99b, hsa-miR-146a,hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p in the CSF of the subjectcompared to the reference level indicates that the subject will have anincreased rate of disease progression. In some embodiments, an increasein the rate of disease progression is an increased rate of onset of oneor more symptoms of ALS, an increase in the worsening of one or moresymptoms of ALS, an increase in the frequency of one or more symptoms ofALS, an increase in the duration of one or more symptoms of ALS, or adecrease in the longevity of the subject.

Also provided are methods of selecting a subject for treatment ofamyotrophic lateral sclerosis (ALS) that include: determining a level ofone or more microRNAs selected from the group consisting of:hsa-miR-19b, hsa-miR-106b, hsa-miR-30b, hsa-miR-21, hsa-miR-142-5p,hsa-miR-27a, hsa-miR-16, hsa-miR-374a, hsa-miR-374b, hsa-miR-101,hsa-miR-340, hsa-miR-30e, hsa-miR-29c, hsa-miR-29a, hsa-miR-223,hsa-miR-26a, hsa-miR-26b, hsa-miR-24, hsa-miR-181a, hsa-miR-103,hsa-miR-155, hsa-miR-532-3p, hsa-miR-518f, hsa-miR-206, hsa-miR- 204,hsa-miR-137, hsa-miR-453, hsa-miR-146a, hsa-miR-603, hsa-miR-1297,hsa-miR-192, hsa-miR-526a, hsa-miR-615-5p, hsa-miR-655, hsa-miR-450b-5p,hsa-miR-548b-3p, hsa-miR-584, hsa-miR-548f, hsa-miR-300, hsa-miR-302c,hsa-miR-328, hsa-miR-421, hsa-miR-580, hsa-miR-15b, and hsa-miR-19a in aCD14⁺CD16⁻ monocyte from the subject; comparing the level of the one ormore microRNAs in a CD14⁺CD16⁻ monocyte from the subject to a referencelevel of the one or more microRNAs; and selecting a subject having anincrease in the level of one or more of hsa-miR-19b, hsa-miR-106b,hsa-miR-30b, hsa-miR-21, hsa-miR-142-5p, hsa-miR-27a, hsa-miR-16,hsa-miR-374a, hsa-miR-374b, hsa-miR-101, hsa-miR-340, hsa-miR-30e,hsa-miR-29c, hsa-miR-29a, hsa-miR-223, hsa-miR-26a, hsa-miR-26b,hsa-miR-24, hsa-miR-181a, hsa-miR-103, hsa-miR-155, hsa-miR-532-3p,hsa-miR-15b, and hsa-miR-19a and/or a decrease in the level of one ormore of hsa-miR-518f, hsa-miR-206, hsa-miR-204, hsa-miR-137,hsa-miR-453, hsa-miR-146a, hsa-miR-603, hsa-miR-1297, hsa-miR-192,hsa-miR-526a, hsa-miR-615-5p, hsa-miR-655, hsa-miR-450b-5p,hsa-miR-548b-3p, hsa-miR-584, hsa-miR-548f, hsa-miR-300, hsa-miR-302c,hsa-miR-328, hsa-miR-421, and hsa-miR-580 in a CD14⁺CD16⁻ monocyte ascompared to the reference level for treatment of ALS.

Also provided are methods for selecting a subject for treatment ofamyotrophic lateral sclerosis (ALS) that include: determining a level ofone or more of hsa-miR-27b, hsa-miR-99b, hsa-miR-146a, hsa-miR-150,hsa-miR-328, and hsa-miR-532-3p in cerebrospinal fluid (CSF) in asubject; and comparing the level of one or more of hsa-miR-27b,hsa-miR-99b, hsa-miR-146a, hsa-miR-150, hsa-miR-328, and hsa-miR-532-3pin the CSF of the subject to a reference level of the one or more ofhsa-miR-27b, hsa-miR-99b, hsa-miR-146a, hsa-miR-150, hsa-miR-328, andhsa-miR-532-3p; and selecting a subject having an increase in the levelof one or more of hsa-miR-27b, hsa-miR-99b, hsa-miR-146a, hsa-miR-150,hsa-miR-328, and hsa-miR-532-3p in the CSF compared to the referencelevel for treatment of ALS.

Also provided are methods for selecting a subject for treatment offamilial amyotrophic lateral sclerosis (ALS) that include determining alevel of hsa-miR-27b and a level of one or more of hsa-miR-99b,hsa-miR-146a, hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p incerebrospinal fluid (CSF) of a subject; and comparing the level ofhsa-miR-27b in the CSF of the subject to a reference level ofhsa-miR-27b, and the level of one or more of hsa-miR-99b, hsa-miR-146a,hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p in the CSF of the subjectto a reference level of one or more of hsa-miR-99b, hsa-miR-146a,hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p; and selecting a subjecthaving an increase in the level of hsa-miR-27b in the CSF compared tothe reference level of hsa-miR-27b, and a decrease or no significantchange in the level of one or more of hsa-miR-99b, hsa-miR-146a,hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p in the CSF compared to thereference level of one or more of hsa-miR-99b, hsa-miR-146a,hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p for treatment of familialALS.

Also provided are methods for selecting a subject for treatment ofsporadic amyotrophic lateral sclerosis (ALS) that include: determining alevel of two or more microRNAs selected from the group consisting ofhsa-miR-27b, hsa-miR-99b, hsa-miR-146a, hsa-miR-150, hsa-miR-328, andhsa-miR-532-3p in the cerebrospinal fluid (CSF) of a subject; comparingthe level of the two or more microRNAs in the CSF of the subject to areference level of the two or more microRNAs; and selecting a subjecthaving an increase in the level of the two or more microRNAs in the CSFcompared to the reference level for treatment of sporadic ALS.

In some embodiments of the methods described herein, the selectedsubject is further administered a treatment for ALS.

Also provided are methods of selecting a subject for participation in aclinical study that include: determining a level of one or moremicroRNAs selected from the group consisting of: hsa-miR-19b,hsa-miR-106b, hsa-miR-30b, hsa-miR-21, hsa-miR-142-5p, hsa-miR-27a,hsa-miR-16, hsa-miR-374a, hsa-miR-374b, hsa-miR-101, hsa-miR-340,hsa-miR-30e, hsa-miR-29c, hsa-miR-29a, hsa-miR-223, hsa-miR-26a,hsa-miR-26b, hsa-miR-24, hsa-miR-181a, hsa-miR-103, hsa-miR-155,hsa-miR-532-3p, hsa-miR-518f, hsa-miR-206, hsa-miR-204, hsa-miR-137,hsa-miR-453, hsa-miR-146a, hsa-miR-603, hsa-miR-1297, hsa-miR-192,hsa-miR-526a, hsa-miR-615-5p, hsa-miR-655, hsa-miR-450b-5p,hsa-miR-548b-3p, hsa-miR-584, hsa-miR-548f, hsa-miR-300, hsa-miR-302c,hsa-miR-328, hsa-miR-421, hsa-miR-580, hsa-miR-15b, and hsa-miR-19a in aCD14⁺CD16⁻ monocyte from the subject; comparing the level of the one ormore microRNAs in a CD14⁺CD16⁻ monocyte from the subject to a referencelevel of the one or more microRNAs; and selecting a subject having anincrease in the level of one or more of hsa-miR-19b, hsa-miR-106b,hsa-miR-30b, hsa-miR-21, hsa-miR-142-5p, hsa-miR-27a, hsa-miR-16,hsa-miR-374a, hsa-miR-374b, hsa-miR-101, hsa-miR-340, hsa-miR-30e,hsa-miR-29c, hsa-miR-29a, hsa-miR-223, hsa-miR-26a, hsa-miR-26b,hsa-miR-24, hsa-miR-181a, hsa-miR-103, hsa-miR-155, hsa-miR-532-3p,hsa-miR-15b, and hsa-miR-19a and/or a decrease in the level of one ormore of hsa-miR-518f, hsa-miR-206, hsa-miR-204, hsa-miR-137,hsa-miR-453, hsa-miR-146a, hsa-miR-603, hsa-miR-1297, hsa-miR-192,hsa-miR-526a, hsa-miR-615-5p, hsa-miR-655, hsa-miR-450b-5p,hsa-miR-548b-3p, hsa-miR-584, hsa-miR-548f, hsa-miR-300, hsa-miR-302c,hsa-miR-328, hsa-miR-421, and hsa-miR-580 in a CD14⁺CD16⁻ monocyte ascompared to the reference level for participation in a clinical study.

Also provided are methods of selecting a subject for participation in aclinical study that include: determining a level of one or moremicroRNAs selected from the group consisting of hsa-miR-27b,hsa-miR-99b, hsa-miR-146a, hsa-miR-150, hsa-miR-328, and hsa-miR-532-3pin the cerebrospinal fluid (CSF) of a subject; comparing the level ofthe one or more microRNAs in the CSF of the subject to a reference levelof the one or more microRNAs; and selecting a subject having an increasein the level of the one or more microRNAs in the CSF compared to thereference level for participation in a clinical study.

Also provided are methods of determining the efficacy of treatment ofamyotrophic lateral sclerosis in a subject that include: determining alevel of one or more microRNAs selected from the group consisting of:hsa-miR-19b, hsa-miR-106b, hsa-miR-30b, hsa-miR-21, hsa-miR-142-5p,hsa-miR-27a, hsa-miR-16, hsa-miR-374a, hsa-miR-374b, hsa-miR-101,hsa-miR-340, hsa-miR-30e, hsa-miR-29c, hsa-miR-29a, hsa-miR-223,hsa-miR-26a, hsa-miR-26b, hsa-miR-24, hsa-miR-181a, hsa-miR-103,hsa-miR-155, hsa-miR-532-3p, hsa-miR-518f, hsa-miR-206, hsa-miR-204,hsa-miR-137, hsa-miR-453, hsa-miR-146a, hsa-miR-603, hsa-miR-1297,hsa-miR-192, hsa-miR-526a, hsa-miR-615-5p, hsa-miR-655, hsa-miR-450b-5p,hsa-miR-548b-3p, hsa-miR-584, hsa-miR-548f, hsa-miR-300, hsa-miR-302c,hsa-miR-328, hsa-miR-421, hsa-miR-580, hsa-miR-15b, and hsa-miR-19a in aCD14⁺CD16⁻ monocyte from the subject at a first time point; determininga level of the one or more microRNAs in a CD14⁺/CD16⁻ monocyte from thesubject at a second time point following administration of at least onedose of a treatment; and comparing the level of the one or moremicroRNAs at the first time point to the level of the one or moremicroRNAs at the second time point; whereby a decrease in the level ofone or more of hsa-miR-19b, hsa-miR-106b, hsa-miR-30b, hsa-miR-21,hsa-miR-142-5p, hsa-miR-27a, hsa-miR-16, hsa-miR-374a, hsa-miR-374b,hsa-miR-101, hsa-miR-340, hsa-miR-30e, hsa-miR-29c, hsa-miR-29a,hsa-miR-223, hsa-miR-26a, hsa-miR-26b, hsa-miR-24, hsa-miR-181a,hsa-miR-103, hsa-miR-155, hsa-miR-532-3p, hsa-miR-15b, and hsa-miR-19aand/or an increase in the level of one or more of hsa-miR-518f,hsa-miR-206, hsa-miR-204, hsa-miR-137, hsa-miR-453, hsa-miR-146a,hsa-miR-603, hsa-miR-1297, hsa-miR-192, hsa-miR-526a, hsa-miR-615-5p,hsa-miR-655, hsa-miR-450b-5p, hsa-miR-548b-3p, hsa-miR-584,hsa-miR-548f, hsa-miR-300, hsa-miR-302c, hsa-miR-328, hsa-miR-421, andhsa-miR-580 at the second time point as compared to the level(s) at thefirst time point indicates that the treatment was effective in thesubject.

Also provided are methods of determining the efficacy of treatment ofamyotrophic lateral sclerosis (ALS) in a subject that include:determining a level of one or more of hsa-miR-27b, hsa-miR-99b,hsa-miR-146a, hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p incerebrospinal fluid of the subject at a first time point; determining alevel of one or more of hsa-miR-27b, hsa-miR-99b, hsa-miR-146a,hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p in CSF of the subject at asecond time point following administration of at least one dose of atreatment; and comparing the level of one or more of hsa-miR-27b,hsa-miR-99b, hsa-miR-146a, hsa-miR-150, hsa-miR-328, and hsa-miR-532-3pat the first time point to the level of one or more of hsa-miR-27b,hsa-miR-99b, hsa-miR-146a, hsa-miR-150, hsa-miR-328, and hsa-miR-532-3pat the second time point; whereby a decrease in the level of one or moreof hsa-miR-27b, hsa-miR-99b, hsa-miR-146a, hsa-miR-150, hsa-miR-328, andhsa-miR-532-3p at the second time point as compared to the level(s) atthe first time point indicates that the treatment was effective in thesubject.

In some embodiments of any of the methods described herein, thereference level is a threshold level. In some embodiments, the referencelevel is a level found in a CD14⁺CD16⁻ monocyte (e.g., a peripheral orblood-derived monocyte) from a control subject. In some embodiments, thereference level is a level found in the CSF of a control subject.

Some embodiments of the methods described herein further includeobtaining a biological sample (e.g., a sample containing blood, plasma,serum, or cerebrospinal fluid) containing a CD14⁺CD16⁻ monocyte from thesubject. In some embodiments, the method further comprises purifying aCD14⁺CD16⁻ monocyte from the biological sample.

Some embodiments of the methods described herein further includeobtaining a sample containing CSF from the subject.

In some embodiments of any of the methods described herein, the microRNAor the one or more microRNA is a precursor microRNA. In some embodimentsof any of the methods described herein, the microRNA or the one or moremicroRNA is a mature microRNA.

Also provided are methods of treating amyotrophic lateral sclerosis(ALS) in a subject that include administering to a subject having ALS atleast one antagomir comprising a sequence that is complementary to acontiguous sequence (e.g., a contiguous sequence of at least 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25nucleotides, preferably at least 7 or 8 nucleotides) present in any oneof hsa-miR-19b, hsa-miR-106b, hsa-miR-30b, hsa-miR-21, hsa-miR-142-5p,hsa-miR-27a, hsa-miR-16, hsa-miR-374a, hsa-miR-374b, hsa-miR-101,hsa-miR-340, hsa-miR-30e, hsa-miR-29c, hsa-miR-29a, hsa-miR-223,hsa-miR-26a, hsa-miR-26b, hsa-miR-24, hsa-miR-181a, hsa-miR-103,hsa-miR-155, hsa-miR-532-3p, hsa-miR-27b, hsa-miR-99b, hsa-miR-146a,hsa-miR-150 hsa-miR-328, has-miR-19a, hsa-miR-15b, hsa-miR-15b, andhsa-miR-19a.

Also provided are methods of treating amyotrophic lateral sclerosis(ALS) in a subject that include administering to a subject having ALS atleast one inhibitory nucleic acid comprising a sequence that iscomplementary to a contiguous sequence present in hsa-miR-155. In someembodiments, the at least one inhibitory nucleic acid is an antagomir(e.g., an antagomir contains or has a sequence of SEQ ID NO: 262). Insome embodiments, the at least one inhibitory nucleic acid is anantisense oligonucleotide. In some embodiments, the at least oneinhibitory nucleic acid is a ribozyme. In some embodiments, the at leastone inhibitory inhibitory nucleic acid is injected into thecerebrospinal fluid of a subject (e.g., intracranial injection orintrathecal injection). In some embodiments, the at least one inhibitorynucleic acid is complexed with one or more cationic polymers and/orcationic lipids. In some embodiments, the inhibitory nucleic acid isdelivered using a lentivirus vector.

Also provided are methods of using at least one antagomir comprising asequence that is complementary to a contiguous sequence present in anyone of hsa-miR-155, hsa-miR-19b, hsa-miR-106b, hsa-miR-30b, hsa-miR-21,hsa-miR-142-5p, hsa-miR-27a, hsa-miR-16, hsa-miR-374a, hsa-miR-374b,hsa-miR-101, hsa-miR-340, hsa-miR-30e, hsa-miR-29c, hsa-miR-29a,hsa-miR-223, hsa-miR-26a, hsa-miR-26b, hsa-miR-24, hsa-miR-181a,hsa-miR-103, hsa-miR-532-3p, hsa-miR-27b, hsa-miR-99b, hsa-miR-146a,hsa-miR-150, hsa-miR-328, hsa-miR-15b, and hsa-miR-19a in themanufacture of a medicament for treating amyotrophic lateral sclerosisin a subject. Also provided herein are antagomirs comprising a sequencethat is complementary to a contiguous sequence present in any one ofhsa-miR-155, hsa-miR-19b, hsa-miR-106b, hsa-miR-30b, hsa-miR-21,hsa-miR-142-5p, hsa-miR-27a, hsa-miR-16, hsa-miR-374a, hsa-miR-374b,hsa-miR-101, hsa-miR-340, hsa-miR-30e, hsa-miR-29c, hsa-miR-29a,hsa-miR-223, hsa-miR-26a, hsa-miR-26b, hsa-miR-24, hsa-miR-181a,hsa-miR-103, hsa-miR-532-3p, hsa-miR-27b, hsa-miR-99b, hsa-miR-146a,hsa-miR-150, hsa-miR-328, hsa-miR-15b, and hsa-miR-19a for use intreating amyotrophic lateral sclerosis in a subject.

Also provided are methods of using at least one inhibitory nucleic acid(e.g., an antagomir) comprising a sequence that is complementary to acontiguous sequence present in hsa-miR-155 in the manufacture of amedicament for treating amyotrophic lateral sclerosis in a subject. Alsoprovided herein are inhibitory nucleic acids (e.g., antagomirs)containing a sequence that is complementary to a contiguous sequencepresent in hsa-miR-155 for use in treating amyotrophic lateral sclerosisin a subject.

As used herein, “RNA” refers to a molecule comprising at least one ormore ribonucleotide residues. A “ribonucleotide” is a nucleotide with ahydroxyl group at the 2′ position of a beta-D- ribofuranose moiety. Theterm RNA, as used herein, includes double-stranded RNA, single- strandedRNA, isolated RNA, such as partially purified RNA, essentially pure RNA,synthetic RNA, recombinantly-produced RNA, as well as altered RNA thatdiffers from naturally-occurring RNA by the addition, deletion,substitution and/or alteration of one or more nucleotides. Nucleotidesof the RNA molecules can also comprise non-standard nucleotides, such asnon-naturally occurring nucleotides or chemically synthesizednucleotides or deoxynucleotides.

A “mature microRNA” (mature miRNA) typically refers to a single-strandedRNA molecules of about 21-23 nucleotides in length, which regulates geneexpression. miRNAs are encoded by genes from whose DNA they aretranscribed, but miRNAs are not translated into protein; instead eachprimary transcript (pri-miRNA) is processed into a short stem-loopstructure (precursor microRNA) before undergoing further processing intoa functional mature miRNA. Mature miRNA molecules are partiallycomplementary to one or more messenger RNA (mRNA) molecules, and theirmain function is to down-regulate gene expression. As used throughout,the term “microRNA” or “miRNA” includes both mature microRNA andprecursor microRNA.

As used herein, the term “inflammatory marker” refers to any of theproteins or mRNAs listed in Tables 20 and 21. The proteins and mRNAslisted in Tables 20 and 21 have been implicated for a role ininflammation. Methods for detecting the levels or activity of theinflammatory markers are known in the art. Additional methods fordetecting the levels or activity of the inflammatory markers aredescribed herein.

By the term “reference level” is meant a control level of one of themicroRNAs listed in Tables 1-19 or one of the inflammatory markerslisted in Tables 20 and 21. A reference level may represent a thresholdlevel of a specific microRNA or inflammatory marker. A reference levelmay also be a level of a particular microRNA or inflammatory markerpresent in the cerebrospinal fluid or in a monocyte (e.g., a CD14⁺CD16⁻or CD14⁺CD16⁺ monocyte (e.g., a peripheral or blood-derived monocyte))from a healthy subject (e.g., a subject that does not present with twoor more symptoms of a neurodegenerative disorder, a subject that has notbeen diagnosed with a neurodegenerative disorder, and/or a subject thathas no family history of neurodegenerative disease).

By the term “increase” is meant an observable, detectable, orsignificant increase in a level as compared to a reference level or alevel measured at an earlier or later time point in the same subject.

By the term “decrease” is meant an observable, detectable, orsignificant decrease in a level as compared to a reference level or alevel measured at an earlier or later time point in the same subject.

By the term “neurodegenerative disorder” is meant a neurologicaldisorder characterized by a progressive loss of neuronal function andstructure, and neuron death. Non-limiting examples of neurodegenerativedisorders include Parkinson's disease (PD), Alzheimer's disease (AD),Huntington's disease (HD), brain stroke, brain tumors, cardiac ischemia,age-related macular degeneration (AMD), retinitis pigmentosa (RP),amyotrophic lateral sclerosis (ALS, e.g., familial ALS and sporadicALS), and multiple sclerosis (MS). Methods for diagnosing aneurodegenerative disorder are described herein. Additional methods fordiagnosing a neurodegenerative disorder are known in the art.

By the term “inhibitory RNA” is meant a nucleic acid molecule thatcontains a sequence that is complementary to a target nucleic acid(e.g., a target microRNA or target inflammatory marker, e.g., any of themicroRNAs listed in Tables 1, 3, 5, 7, 9, 11, 12, 14, 16, or 18, or anyof the inflammatory markers listed in Table 21) that mediates a decreasein the level or activity of the target nucleic acid (e.g., activity inCD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte). Non-limiting examples of inhibitoryRNAs include interfering RNA, shRNA, siRNA, ribozymes, antagomirs, andantisense oligonucleotides. Methods of making inhibitory RNAs aredescribed herein. Additional methods of making inhibitory RNAs are knownin the art.

As used herein, “an interfering RNA” refers to any double stranded orsingle stranded RNA sequence, capable—either directly or indirectly(i.e., upon conversion)—of inhibiting or down regulating gene expressionby mediating RNA interference. Interfering RNA includes but is notlimited to small interfering RNA (“siRNA”) and small hairpin RNA(“shRNA”). “RNA interference” refers to the selective degradation of asequence-compatible messenger RNA transcript.

As used herein “an shRNA” (small hairpin RNA) refers to an RNA moleculecomprising an antisense region, a loop portion and a sense region,wherein the sense region has complementary nucleotides that base pairwith the antisense region to form a duplex stem. Followingpost-transcriptional processing, the small hairpin RNA is converted intoa small interfering RNA by a cleavage event mediated by the enzymeDicer, which is a member of the RNase III family.

A “small interfering RNA” or “siRNA” as used herein refers to any smallRNA molecule capable of inhibiting or down regulating gene expression bymediating RNA interference in a sequence specific manner. The small RNAcan be, for example, about 18 to 21 nucleotides long.

As used herein, an “antagomir” refers to a small synthetic RNA havingcomplementarity to a specific microRNA target, optionally with eithermispairing at the cleavage site or one or more base modifications toinhibit cleavage.

As used herein, the phrase “post-transcriptional processing” refers tomRNA processing that occurs after transcription and is mediated, forexample, by the enzymes Dicer and/or Drosha.

By the phrase “risk of developing disease” is meant the relativeprobability that a subject will develop a neurodegenerative disorder inthe future as compared to a control subject or population (e.g., ahealthy subject or population). Provided herein are methods fordetermining a subject's risk of developing a neurodegenerative diseasein the future that include determining the level of one or more of themicroRNAs listed in Tables 1-19 and/or one or more of the inflammatorymarkers listed in Tables 20-21.

By the phrase “rate of disease progression” is meant one or more of therate of onset of symptoms of a neurodegenerative disorder in a subject,the rate of the increasing intensity (worsening) of symptoms of aneurodegenerative disorder in a subject, the frequency of one or moresymptoms of a neurodegenerative disorder in a subject, the duration ofone or more symptoms of a neurodegenerative disorder in a subject, orthe longevity of subject. For example, an increased rate of diseaseprogression can include one or more of: an increased rate of onset ofsymptoms of a neurodegenerative disorder in a subject, an increasedfrequency of one or more symptoms of a neurodegenerative disorder in asubject, an increase in the duration of one or more symptoms of aneurodegenerative disorder in a subject, or a decrease in the longevityof a subject. Methods of predicting the rate of disease progression in asubject having a neurodegenerative disorder are described herein.

By the term “purifying” is meant a partial isolation of a substance fromits natural environment (e.g., partial removal of contaminatingbiomolecules or cells). For example, a monocyte (e.g., a CD14⁺CD16⁻ orCD14⁺CD16⁺ monocyte) can be purified from other cell types present in asample of peripheral blood (e.g., using fluorescence-assisted cellsorting).

The term “treating” includes reducing the number of symptoms or reducingthe severity, duration, or frequency of one or more symptoms of disease(e.g., a neurodegenerative disease) in a subject. The term treating canalso include reducing the risk of developing a neurodegenerativedisorder in a subject, delaying the onset of symptoms of aneurodegenerative disorder in a subject, or increasing the longevity ofa subject having a neurodegenerative disorder.

By the term “cationic polymer” is meant a polymeric material that ispositively-charged at a physiological pH (e.g., a pH of approximately6.5 to 8.0) that is capable of condensing nucleic acids intonanoparticles. Non-limiting examples of cationic polymers includepoly-L-lysine and poly(ethylenimine). Additional examples of cationicpolymers are known in the art.

By the term “cationic lipids” is meant a lipid that has at least onepositive charge at a physiological pH (e.g., a pH of approximately 6.5to 8.0) that is able to form a complex with a nucleic acid. Non-limitingexamples of cationic lipids include 1,2-dioleoyl-3-trimethylammoniumpropone (DOTAP), N-methyl-4-(dioleyl)methylpyridinium, and3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol. Additionalexamples of cationic lipids are known in the art and are commerciallyavailable (e.g., Lipofectamine™ 2000; Life Technologies Corporation,Carlsbad, Calif.).

Other definitions appear in context throughout this disclosure. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Methods and materials are describedherein for use in the present invention; other, suitable methods andmaterials known in the art can also be used. The materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, sequences, database entries,and other references mentioned herein are incorporated by reference intheir entirety. In case of conflict, the present specification,including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a volcano plot of significantly dysregulated microRNAs inCD39⁺ microglia in SOD^(G93A) mice compared to the expression ofmicroRNAs in CD39+ microglia from non- transgenic litermates at apresymptomatic (60 days) time point (Presymptomatic), at the time ofonset of symptoms (Onset), and at the end-stage of the disease(End-Stage). The x-axis represents changes in expression (logy-foldchange based on ddCT values) and the y-axis shows the statisticalsignificance of the change in log-odds.

FIG. 1B is a Venn diagram of significantly dysregulated microRNAs inCD39⁺ microglia in SOD^(G93A) mice compared to the expression of themicroRNAs in CD39⁺ microglia from non-transgenic litermates across alldisease stages. The numbers represent significantly dysregulatedmicroRNAs at each disease stage.

FIG. 1C is a summary of significantly dysregulated microRNAs in CD39+microglia in SOD^(G93A) mice compared to the expression of the microRNAsin CD39⁺ microglia from non-transgenic litermates. These data werevalidated in singleplex TaqMan PCR.

FIG. 2A is a volcano plot of significantly dysregulated microRNAs inLy6C^(Hi) monocytes in SOD^(G93A) mice compared to the expression of themicroRNAs in Ly6C^(Hi) monocytes from non-transgenic litermates at apresymptomatic (60 days) time point (Presymptomatic), at the time ofonset of symptoms (Onset), and at the end-stage of disease (End-Stage).The x-axis represents changes in expression (logy-fold change based onddCT values) and the y-axis shows statistical significance of the changein log-odds.

FIG. 2B is a Venn diagram of significantly dysregulated microRNAs inLy6C^(Hi) monocytes in SOD^(G93A) mice compared to the expression of themicroRNAs in Ly6C^(Hi) monocytes from non-transgenic litermates acrossall disease stages (presymptomatic, onset of symptoms, and the end-stageof disease). The numbers represent significantly dysregulated microRNAsat each disease stage.

FIG. 2C is a summary of significantly dysregulated microRNAs inLy6C^(Hi) monocytes in SOD^(G93A) mice compared to the expression of themicroRNAs in Ly6C^(Hi) monocytes from non-transgenic litermates. Thesedata were validated in singleplex TaqMan PCR.

FIG. 3A is a volcano plot of significantly dysregulated microRNAs inLy6C^(Low) monocytes in SOD^(G93A) mice compared to the expression ofthe microRNAs in Ly6C^(Low) monocytes from non-transgenic litermates ata presymptomatic (60 days) time point, at the time of onset of symptoms(Onset), and at the end-stage of disease (End-Stage). The x-axisrepresents changes in expression (logy-fold change based on ddCT values)and the y-axis shows statistical significance of the change in log-odds.

FIG. 3B is a Venn diagram of significantly dysregulated microRNAs inLy6C^(Low) monocytes in SOD^(G93A) mice compared to the expression ofthe microRNAs in Ly6C^(Low) monocytes from non-transgenic litermatesacross all disease stages. The numbers represent significantlydysregulated microRNAs at each disease stage.

FIG. 3C is a summary of significantly dysregulated microRNAs inLy6C^(Low) monocytes in SOD^(G93A) mice compared to the expression ofthe microRNAs in Ly6C^(Low) monocytes from non-transgenic litermates.These data were validated in singleplex TaqMan PCR.

FIG. 4 is a graph and a table showing the results of Ingenuity pathwayanalysis of the 32 dysregulated microRNAs in Ly6C^(Hi) monocytes (ascompared to non-transgenic litermate controls) across all disease stagesin SOD1 mice. The graph shows patterns observed in skeletal diseases,muscular diseases, and myopathic disorders. FIG. 5 is a heatmap showingthe nCounter expression profiling of blood-derived CD14⁺CD16⁻ monocytesfor 664 microRNAs in sporadic ALS (8 subjects) and relapsing-remittingmultiple sclerosis (8 subjects) compared to the expression of themicroRNAs in CD14⁺CD16⁻ monocytes from healthy controls (8 subjects).The heatmap shows the results of analysis of variance (ANOVA) usingDunnett's post hoc test (P<0.01). The microRNAs upregulated ordownregulated in CD14⁺CD16⁻ monocytes from ALS subjects (as compared toexpression of these microRNAs in CD14⁺CD16⁻ monocytes from healthycontrols) are indicated. Each row of the heatmap represents anindividual gene and each column an individual.

FIG. 6 is a heatmap showing the nCounter expression profiling ofblood-derived CD14⁺CD16⁻ monocytes for 664 microRNAs in sporadic ALS (8subjects) and relapsing-remitting multiple sclerosis (8 subjects)compared to the expression of these microRNAs in CD14⁺CD16⁻ monocytesfrom healthy controls (8 subjects). The heatmap shows the results ofANOVA using Dunnett's post hoc test (P<0.01). The microRNAs upregulatedor downregulated in CD14⁺CD16⁻ monocytes from MS subjects (as comparedto the expression of the microRNAs in CD14⁺CD16⁻ monocytes from healthycontrols) are indicated. Each row of the heatmap represents anindividual gene and each column an individual.

FIG. 7A is a Venn diagram of the unique or similar dysregulated(upregulated or downregulated) microRNAs in CD14⁺CD16⁻ monocytes fromALS and MS subjects as compared to the expression of the microRNAs inCD14⁺CD16⁻ monocytes from healthy controls.

FIG. 7B is a volcano plot showing the significantly dysregulatedmicroRNAs in CD14⁺CD16⁻ monocytes from ALS subjects as compared to theexpression of the microRNAs in CD14⁺CD16⁻ monocytes from healthycontrols.

FIG. 7C is a volcano plot showing the significantly dysregulatedmicroRNAs in CD14⁺CD16⁻ monocytes from MS subjects as compared to theexpression of the microRNAs in CD14⁺CD16⁻ monocytes from healthycontrols.

FIG. 8 is a summary of the significantly dysregulated microRNAs inCD14⁺CD16⁻ monocytes from ALS and MS subjects compare d to theexpression of the microRNAs in CD14⁺CD16⁻ monocytes from healthycontrols. The bars show the relative expression of dysregulatedmicroRNAs in CD14⁺CD16⁻ monocytes from ALS and MS subjects compared tothe expression of the microRNAs in CD14⁺CD16⁻ monocytes from healthycontrols.

FIG. 9 is a set of six graphs showing the expression of six differentmicroRNAs in CD14⁺CD16⁻ monocytes from healthy subjects (8 subjects) andsubjects having ALS (11 subjects) (as determined by real-time PCR). Atwo-tiled Mann-Whitney t-test was used to calculate the P values (*,P<0.05; **, P<0.01; ***, P<0.001).

FIG. 10A is two graphs showing the clinical scoring (forced vitalcapacity (FVC) score and Functional Rating Scale (FRS)) of eightdifferent ALS patients. A comparison of the microRNA expression inCD14⁺CD16⁻ monocytes from these eight patients to microRNA expression inCD14⁺CD16⁻ monocytes from healthy controls and MS subjects are shown inFIG. 10C.

FIG. 10B is a list of the eight different ALS patients described inFIGS. 10A and 10C.

FIG. 10C is twenty graphs showing the expression of twenty upregulatedmicroRNAs in CD14⁺CD16⁻ monocytes from sporadic ALS subjects (8subjects), healthy subjects, and subjects having MS (determined usingreal-time PCR). A two-tiled Mann-Whitney t-test was used to calculatethe P values.

FIG. 11 is four graphs showing the real-time PCR analysis of theexpression of four upregulated microRNAs in CD14⁺CD16⁻ monocytes fromsporadic ALS (n=11) subjects as compared to healthy controls (n=8), andsubjects with MS (n=8). The data shown were generated using one-wayANOVA and the Dunett's multiple comparison test (***, p<0.001). 4

FIG. 12A is two graphs showing the clinical scoring (forced vitalcapacity (FVC) score and Functional Rating Scale (FRS)) of eightdifferent ALS patients. A comparison of the microRNA expression inCD14⁺CD16⁻ monocytes from these eight patients to microRNA expression inCD14⁺CD16⁻ monocytes from healthy controls and MS subjects are shown inFIG. 10C.

FIG. 12B is a list of the eight different ALS patients described inFIGS. 12A and 12C.

FIG. 12C is twenty graphs showing the expression of twenty downregulatedmicroRNAs in CD14⁺CD16⁻ monocytes from sporadic ALS subjects as comparedto healthy subjects, and subjects having MS-relapsing remitting (MS-RR)(determined using real-time PCR). A two-tiled Mann-Whitney t-test wasused to calculate the P values (*, P<0.05; **, P<0.01; ***, P<0.001).

FIG. 13 is eight graphs showing the expression of eight upregulatedmicroRNAs in CD14⁺CD16⁻ monocytes from sporadic ALS and MS-RR ascompared to healthy subjects, subjects (8 subjects in each group)(determined using real-time PCR). The data shown were generated usingone-way ANOVA and the Dunett's multiple comparison test (**, P<0.01;***, p<0.001).

FIG. 14 is five different graphs showing the expression of fiveupregulated microRNAs in CD14⁺CD16⁻ monocytes from MS-RR subjects ascompared to healthy subjects and subjects having ALS (8 subjects)(determined using real-time PCR). A two-tiled Mann-Whitney t-test wasused to calculate the P values (*, P<0.05; **, P<0.01; ***, P<0.001).

FIG. 15 is five graphs showing the expression of five downregulatedmicroRNAs in CD14⁺CD16⁻ monocytes from MS-RR subjects as compared tohealthy subjects, and subjects having sporadic ALS (determined byreal-time PCR). A two-tiled Mann-Whitney t-test was used to calculatethe P values (*, P<0.05).

FIG. 16 is six graphs showing the expression of six upregulatedmicroRNAs in the cerebrospinal fluid (CSF) from healthy subjects,subjects with familial ALS (n=5), and subjects with sporadic ALS (n=10).The data were analyzed using ANOVA with Bonfferoni's multiple comparisontest. *, p<0.05; **, p<0.01; and ***, p<0.001.

FIG. 17 is a heatmap showing the nCounter expression profiles of 179inflammation related genes (“inflammatory marker genes”) in CD14⁺CD16⁻monocytes from ALS subjects (n=8) and MS subjects (n=11) compared to thelevels of these inflammatory marker genes in CD14⁺CD16⁻ monocytes fromhealthy controls (n=10). Data analysis was performed using ANOVA withDunnett's post hoc test (p<0.01). Each row of the heatmap represents anindividual gene and each column represents an individual subject.

FIG. 18A is two volcano plots showing the significantly dysregulatedinflammatory marker genes in CD14⁺CD16⁻ monocytes from ALS subjects(left graph) and MS subjects (right graph) compared to the level of theinflammatory marker genes in CD14⁺CD16⁻ monocytes from healthy controls.

FIG. 18B is a summary of the significantly dysregulated inflammatorymarker genes in CD14⁺CD16⁻ monocytes from ALS and MS subjects comparedto the level of the inflammatory marker genes in CD14⁺CD16⁻ monocytesfrom healthy controls. The bars show the relative expression ofdysregulated inflammatory marker genes in CD14⁺CD16⁻ monocytes from ALSand MS subjects compared to the expression of these genes in CD14⁺CD16⁻monocytes from healthy controls.

FIG. 19 is eight graphs showing the expression of eight differentmicroRNAs in CD14⁺CD16⁺ monocytes from healthy controls (n=8) and ALSsubjects (n=11) (determined using real-time PCR). The data were analyzedusing the two-tiled Mann-Whitney t-test (*, P<0.05).

FIG. 20A is a heatmap showing the nCounter expression profiles ofmicroRNAs in CD14⁺CD16⁺ monocytes from ALS subjects (n=8) and MSsubjects (n=8) compared to the expression of these microRNAs inCD14⁺CD16⁺ monocytes from healthy controls (n=8). Data analysis wasperformed using ANOVA with Dunnett's post hoc test (p<0.01). Each row ofthe heatmap represents an individual gene and each column represents anindividual subject. MicroRNAs upregulated or downregulated in CD14⁺CD16⁺monocytes from ALS subjects relative to CD14⁺CD16⁺ monocytes fromhealthy subjects are indicated.

FIG. 20B is a heatmap showing the nCounter expression profiles ofmicroRNAs in CD14⁺CD16⁺ monocytes from ALS subjects (n=8) and MSsubjects (n=8) compared to the expression of the microRNAs in CD14⁺CD16⁺monocytes from healthy controls (n=8). Data analysis was performed usingANOVA with Dunnett's post hoc test (p<0.01). Each row of the heatmaprepresents an individual gene and each column represents an individualsubject. MicroRNAs upregulated or downregulated in CD14⁺CD16⁺ monocytesfrom MS subjects relative to the expression of the microRNAs inCD14⁺CD16⁺ monocytes from healthy subjects are indicated.

FIG. 20C is a summary of the significantly dysregulated microRNAs inCD14⁺CD16⁻ monocytes in ALS and MS subjects compared to the expressionof the microRNAs in CD14⁺CD16⁺ monocytes from healthy controls. The barsshow the relative expression of dysregulated microRNAs in CD14⁺CD16⁺monocytes from ALS and MS subjects compared to the expression of themicroRNAs in CD14⁺CD16⁺ monocytes in healthy controls.

FIG. 21A is nCounter expression profiles of 179 inflammatory markergenes in Ly6C^(Hi) spleen-derived monocyte subsets from SOD1^(G93A) micecompared to the same cells non-transgenic (Tg) litermates atpresymptomatic (60d), onset (defined by body weight loss), and end-stageof disease. A heatmap of the ANOVA with Dunett's post hoc test (P<0.01)results showing genes with at least 2-fold altered transcription levelsis shown. Each row of the heatmap represents an individual gene and eachcolumn an individual group in biological triplicates (n=3 arrays foreach group of pool of 4-5 mice at each time point). Non-transgenicreplicates at each disease stage were collapsed and genes hierarchicallyclustered. Gene expression level was normalized against the geometricmean of six house-keeping genes (CLTC, GAPDH, GUSB, HPRT1, PGK1, andTUBBS).

FIG. 21B is nCounter expression profile data showing inflammatory markergenes that are significantly downregulated in Ly6C^(Hi) spleen-derivedmonocyte subsets from SOD1^(G93A) mice compared to the same cells innon-transgenic (Tg) litermates at presymptomatic (60d), onset (definedby body weight loss), and end-stage of disease.

FIG. 21C is a list of the major biological networks activated inLy6C^(Hi) spleen-derived monocytes one month prior to disease onset inSOD1^(G93A) mice.

FIG. 21D shows the nCounter expression profile data of genes upregulatedin spinal cord-derived CD39⁺ microglia from SOD1^(G93A) mice compared tothe same cells from non- transgenic litermates.

FIG. 21E shows the nCounter expression profile data of genesdownregulated in spinal cord-derived CD39⁺ microglia from SOD1^(G93A)mice compared to the same cells from non- transgenic litermates.

FIG. 21F is a list of the major biological pathways activated in CD39⁺microglia the spinal cords of SOD1 mice at the onset of disease

FIG. 21G is a comparative analysis of the significantly upregulatedgenes in CD39 ⁺ microglia from spinal cords of SOD1 mice at the onsetversus CD39⁺ microglia isolated from the brain of the same SOD1 mice.

FIG. 22A is an nCounter expression profile of 184 inflammation-relatedgenes in CD14+CD16⁻ blood monocytes from sporadic ALS (n=11) and MS(n=8) subjects compared to healthy controls (n=10).

FIG. 22B is a graphic showing the fold differences in expression ofsignificantly dysregulated genes in sporadic ALS and MS subjects ascompared to healthy controls. Gene expression level was normalizedagainst the geometric mean of 6 internal reference house-keeping genes(CLTC, GAPDH, GUSB, PGK1, and TUBBS).

FIG. 22C is a graphic showing the principal components analysis (PCA)analysis of the identified dysregulated genes between sporadic ALSsubjects and MS subjects with spatial gene distribution.

FIG. 23A is an nCounter expression profile of blood-sorted CD14⁺CD16⁻monocytes for 511 immune- and 184-inflammation-related genes in sporadicALS (10 subjects), and familial SOD1 ALS (4 subjects) compared tohealthy controls (10 subjects). The profile (heatmap) is an unsupervisedhierarchial clustering (Pearson correlation) that shows thesignificantly dysregulated genes (Nonparametric Kruskal-Wallis test;significance based on false discovery rate (FDR) determined by theBenjamini-Hochberg method; selected FDR limit: 0.05; P<0.01). Each rowof the heatmap represents an individual gene and each column anindividual subject.

FIG. 23B is a graphic showing the fold differences of significantlydysregulated genes in blood-sorted CD14⁺CD16⁻ monocytes from sporadicALC and familial ALS subjects versus healthy controls. Gene expressionlevel was normalized against the geometric mean of 15 internal referencehouse-keeping genes (ABCF1, ALAS1, EEF1G, G6PD, GAPDH, GUSB, HPRT1,OAZ1, POLR1B, POLR2A, PPIA, RPL19, DSHA, TBP, and TUBB).

FIG. 23C is a graphic of the PCA analysis of the identified dysregulatedgenes in blood-sorted CD14⁺CD16⁻ monocytes from sporadic ALS andfamilial ALS subjects vs. healthy controls with spatial genedistribution.

FIG. 24 is a set of eight graphs showing the real-time PCR validation ofeight genes that were the most significantly dysregulated inblood-sorted CD14⁺CD16⁻ monocytes from familial and /or sporadic ALSsubjects compared to blood-sorted CD14⁺CD16⁻ monocytes from healthycontrols. The relative expression in sporadic ALS and familial ALSagainst healthy controls was calculated using the comparative Ct(2-ΔΔCt) method. Gene expression level was normalized against thegeometric mean of three house-keeping genes (GAPDH, TUBB, and GRB2). Thepolymerase chain reactions were run in duplicate for each subject. Thegraphs represent one-way analysis of variance (ANOVA) and the Dunett'smultiple comparison test of significantly dysregulated genes in ALSsubjects.

FIG. 25 is a graphic of the Ingenuity target filter analysis showing thetop 10 miRNA-mRNA interactions in CD14⁺CD16⁻ blood monocytes from ALSsubjects based on the identified significantly dysregulated miRNAs andmRNAs in CD14⁺CD16⁻ blood monocytes from ALS subjects.

FIG. 26 is a table of the results of the microRNA-mRNA target analysisperformed on the data gathered from CD14⁺CD16⁻ blood monocytes from ALSsubjects (IPA; Ingenuity). The results show 32 miRNAs targeting 27mRNAs.

FIG. 27 is a graphic depicting the microRNA-mRNA interactions inblood-sorted CD14⁺CD16⁻ monocytes in ALS. The graphic depicts theresults for the significantly dysregulated miRNA and immune-relatedgenes in blood-sorted CD14⁺CD16⁻ monocytes from ALS subjects. A total of32 miRNAs targeting 27 mRNAs are shown.

FIG. 28A and 28B are two graphs showing the distribution of possiblerandom interactions between 1000 random and non-regulated miRNA-mRNApairs in comparison to the observed putative miRNA-mRNA pairs in 41non-regulated highly expressed miRNAs and the 47 dysregulated genesobserved in splenic Lys6C^(Hi) monocytes from SOD1 mice (FIG. 28A), and64 non-regulated highly expressed miRNAs and the 59 dysregulated genesobserved in CD14⁺CD16⁻ peripheral blood monocytes from ALS subjects(FIG. 28B) (Targetscan 4.1).

FIG. 29 is a table showing the top 20 transcription factors and targetgenes dysregulated in blood-sorted CD14+CD16− monocytes from ALSsubjects (determined using GeneGo pathway analysis), and a graphicshowing the specificity protein-1 (SP1) transcription factor and itstargeted genes in blood-sorted CD14+CD16− monocytes in ALS subjects.

FIG. 30 is a graph of the Kaplan-Meir analysis of the probability ofsurviving for both the SOD1/miR-155^(−/−) and the SOD1/miR-155^(+/−)mice. Mantel-Cox's F-test comparison between groups SOD1/miR-155^(−/−)vs. SOD1/miR-155^(+/−) mice (P<0.0001).

FIG. 31 is graph of the time-to-event analysis for disease neurologiconset (neurological severity score 2). Disease onset was significantlydelayed (P<0.0001) in SOD1/miR-155^(−/−) mice compared to theSOD1/miR-155^(+/−) mice.

FIG. 32 is a graph of the rotarod performance of the SOD1/miR-155^(−/−)(circles) and SOD1/miR-155^(+/−) (triangles) mice as a function of age.**P<0.01; ***P<0.001; by factorial ANOVA and Fisher's LSD post-hoc test.

FIG. 33 is a graph of the weight loss of SOD1/miR-155^(−/−) (circles)and SOD1/miR-155^(+/−) (triangles) mice. Statistical analysis wasperformed using 2-way ANOVA, Bonferri post-hoc test. ***P<0.001.

FIG. 34 is a set of two graphs showing the duration of an early diseasephase (from onset to 5% weight loss) (left graph) and duration of anlater disease phase (from 5% weight loss to end stage) for theSOD1/miR-155^(−/−) and SOD1/miR-155^(+/−) mice.

FIG. 35A shows the fluorescence-activated cell sorting (FACS) analysisdata of spinal cord-derived mononuclear cells stained with 4D4 (residentmicroglia) and CD11b (myeloid cells) in wildtype, SOD1/miR155^(+/+),SOD1/miR155^(−/ +), and SOD1/miR155^(−/−) mice.

FIG. 35B shows the absolute number of microglia (4D4 positive) andmonocyte cells (CD1lb positive) cells per spinal cord in wildtype,SOD1/miR155^(+/+), SOD1/miR155^(−/+), and SOD1/miR155^(−/−) mice.

FIG. 36 is a set of four heat maps of showing the expression ofinflammation-related genes in spinal cord microglia and Ly6C^(Hi)splenic monocytes in WT, SOD1/miR155^(−/+), and SOD1/miR155^(−/−) mice.The heat maps labeled (a) are from animals at end-stage. (Note thatSOD1/miR155^(−/−) mice are still viable and breeding at the end of thestudy, while SOD1/miR^(−/ +) mice experience an onset of symptoms (endstage)). All mice are males C57/B16-SOD1 background. The heat mapslabeled (b) indicate the genes significantly affected by miR155 in SOD1mice.

FIG. 37 is nCounter expression profile data showing the expression ofseveral mouse microRNAs in Ly6CHi spleen-derived monocyte subsets fromwild type, SOD1/miR155^(−/+), SOD1/miR155^(−/−) mice.

FIG. 38 is a heatmap and bar graphs showing the nCounter expressionprofiling of blood-derived CD14⁺CD16⁻ monocytes for microRNAs insporadic ALS (8 subjects) and relapsing-remitting multiple sclerosis (8subjects) compared to the expression of the microRNAs in CD14⁺CD16⁻monocytes from healthy controls (8 subjects). The heatmap shows theresults of analysis of variance (ANOVA) using Dunnett's post hoc test(P<0.01). The microRNAs upregulated or downregulated in CD14⁺CD16⁻monocytes from ALS subjects (as compared to expression of thesemicroRNAs in CD14⁺CD16⁻ monocytes from healthy controls) are indicated.Each row of the heatmap represents an individual gene and each column anindividual.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based, at least in part, on the discovery that specificmicroRNAs and inflammatory markers are dysregulated in CD14⁺CD16⁻monocytes and/or CD14⁺CD16⁻ monocytes (e.g., peripheral or blood-derivedmonocytes) from patients having a neurodegenerative disease, and thatspecific microRNAs are present in increased or decreased levels in thecerebrospinal fluid of patients having a neurodegenerative disorder(e.g., ALS (e.g., sporadic ALS and familial ALS) and MS) compared tohealthy individuals. The invention is also based on the discovery thathsa-miR-155 plays a significant role in the development of disease in amouse model of ALS. In view of this discovery, methods for diagnosing aneurodegenerative disorder, identifying a subject at risk (e.g.,increased risk or decreased risk) of developing a neurodegenerativedisorder, predicting the rate of disease progression in a subject havinga neurodegenerative disorder, selecting a subject for treatment of aneurodegenerative disorder, selecting a treatment for a subject having aneurological disorder, determining the efficacy of treatment of aneurodegenerative disorder, and selecting a subject for participation ina clinical study are provided herein. These methods include measuring alevel of one or more (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10)microRNAs listed in one or more of Tables 1-19 and/or one or moreinflammatory markers listed in Tables 20-21.

Also provided are methods of treating a neurodegenerative disorder(e.g., ALS or MS) that include administering to a subject an agent(e.g., a nucleic acid) that decreases the level or activity of one ormore of the microRNAs listed in Tables 1, 3, 5, 7, 9, 11, 12, 14, 16, or18 (e.g., hsa-miR-155), and/or increases the level or activity of one ormore of the microRNAs listed in Tables 2, 4, 6, 8, 10, 13, 15, 17, or19. Also provided are methods of treating a neurological disorder (e.g.,ALS or MS) that include administering to a subject an agent (e.g., anucleic acid) that decreases the expression (e.g., protein or mRNA)and/or activity of one or more of the inflammatory markers listed inTable 21 and/or increases the expression (e.g., protein or mRNA) and/oractivity of one or more of the genes listed in Table 20.

Also provided are nucleic acids that contain a sequence complementary toa sequence present in any one of the microRNAs listed in Tables 1-19 ora sequence present in an mRNA that encodes any of the genes listed inTables 20 and 21 (e.g., a primer or probe). Also provided are nucleicacids that contain a sequence that is complementary to a sequencepresent in any one of the microRNAs listed in Tables 1, 3, 5, 7, 9, 11,12, 14, 16, or 18 (the target microRNA), or a sequence present in a mRNAencoded by any of the genes listed in Table 21 (the target mRNA), thatdecrease the expression or activity of the target microRNA or targetmRNA (e.g., an inhibitory RNA, e.g., any of the inhibitory nucleic acidsdescribed herein). Also provided are compositions that contain a nucleicacid that includes the sequence of any one of the microRNAs listed inTables 2, 4, 6, 8, 10, 13, 15, 17, and 19 (the target microRNA), or asequence present in an mRNA encoded by any of the genes listed in Table20 (the target mRNA), that increase the expression or activity of thetarget microRNA or target mRNA. Also included are compositions thatcontain at least one antibody that specifically binds to any one of theproteins listed in Table 20 and Table 21. Also included are compositionsthat contain at least one protein listed in Table 20 and Table 21. Alsoprovided are kits that contain one or more of the above nucleic acids,proteins, or antibodies (in any combination).

Neurodegenerative Disorders

Neurodegenerative disorders are a class of neurological diseases thatare characterized by the progressive loss of the structure and functionof neurons and neuronal cell death. Inflammation has been implicated fora role in several neurodegenerative disorders. Progressive loss of motorand sensory neurons and the ability of the mind to refer sensoryinformation to an external object is affected in different kinds ofneurodegenerative disorders. Non-limiting examples of neurodegenerativedisorders include Parkinson's disease, Alzheimer's disease, Huntington'sdisease, amyotrophic lateral sclerosis (ALS, e.g., familial ALS andsporadic ALS), and multiple sclerosis (MS).

A health care professional may diagnose a subject as having aneurodegenerative disorder by the assessment of one or more symptoms ofa neurodegenerative disorder in the subject. Non-limiting symptoms of aneurodegenerative disorder in a subject include difficulty lifting thefront part of the foot and toes; weakness in arms, legs, feet, orankles; hand weakness or clumsiness; slurring of speech; difficultyswallowing; muscle cramps; twitching in arms, shoulders, and tongue;difficulty chewing; difficulty breathing; muscle paralysis; partial orcomplete loss of vision; double vision; tingling or pain in parts ofbody; electric shock sensations that occur with head movements; tremor;unsteady gait; fatigue; dizziness; loss of memory; disorientation;misinterpretation of spatial relationships; difficulty reading orwriting; difficulty concentrating and thinking; difficulty makingjudgments and decisions; difficulty planning and performing familiartasks; depression; anxiety; social withdrawal; mood swings;irritability; aggressiveness; changes in sleeping habits; wandering;dementia; loss of automatic movements; impaired posture and balance;rigid muscles; bradykinesia; slow or abnormal eye movements; involuntaryjerking or writhing movements (chorea); involuntary, sustainedcontracture of muscles (dystonia); lack of flexibility; lack of impulsecontrol; and changes in appetite. A health care professional may alsobase a diagnosis, in part, on the subject's family history of aneurodegenerative disorder. A health care professional may diagnose asubject as having a neurodegenerative disorder upon presentation of asubject to a health care facility (e.g., a clinic or a hospital). Insome instances, a health care professional may diagnose a subject ashaving a neurodegenerative disorder while the subject is admitted in anassisted care facility. Typically, a physician diagnoses aneurodegenerative disorder in a subject after the presentation of one ormore symptoms.

Provided herein are additional methods for diagnosing aneurodegenerative disorder in a subject (e.g., a subject presenting withone or more symptoms of a neurodegenerative disorder or a subject notpresenting a symptom of a neurodegenerative disorder (e.g., anundiagnosed and/or asymptomatic subject). Also provided herein areprognostic methods and methods of treating a neurodegenerative disorderin a subject (e.g., methods of decreasing the rate of onset or theprogression of symptoms (e.g., ataxia) of a neurodegenerative disorderin a subject).

Markers

Any combination of one or more of the markers described herein can beused in any of the methods described herein, e.g., used in methods fordiagnosing a neurodegenerative disorder in a subject, identifying asubject at risk (e.g., increased or decreased risk) of developing aneurodegenerative disorder, predicting the rate of disease progressionin a subject having a neurodegenerative disorder, selecting a subjectfor treatment of a neurodegenerative disorder, determining the efficacyof treatment in a subject having a neurodegenerative disorder, orselecting a subject for participation in a clinical study.

MicroRNA markers increased in monocytes (CD14⁺CD16⁻ or CD14⁺CD16⁺monocytes) or the CSF in subjects having a neurodegenerative disorderrelative to healthy controls (CD14⁺CD16⁻ or CD14⁺CD16⁺ monocytes, or theCSF in healthy controls) are listed in Table 1.

TABLE 1List of microRNAs increased in CD14⁺CD16⁻ monocytes, CD14⁺CD16⁻ monocytes, or CSFfrom patients having neurodegenerative disorders compared to healthy controlsMature miRNA MiRNA sequence Precursor miRNA sequence hsa-miR-19bgugcaaauccaugcaaaacuga CACUGUUCUAUGGUUAGUUUUGCAGGUUUGCAUCCAGCUGUGUGAU(SEQ ID NO: 1) AUUCUGCUGUGCAAAUCCAUGCAAAACUGACUGUGGUAGUGugugcaaauccaugcaaaacuga (SEQ ID NO: 3) (SEQ ID NO: 2)ACAUUGCUACUUACAAUUAGUUUUGCAGGUUUGCAUUUCAGCGUAUAUAUGUAUAUGUGGCUGUGCAAAUCCAUGCAAAACUGAUUGUGAUA AUGU (SEQ ID NO: 4)hsa-miR-106b uaaagugcugacagugcagauCCUGCCGGGGCUAAAGUGCUGACAGUGCAGAUAGUGGUCCUCUCCG (SEQ ID NO: 5)UGCUACCGCACUGUGGGUACUUGCUGCUCCAGCAGG (SEQ ID NO: 6) hsa-miR-30buguaaacauccuacacucagcu ACCAAGUUUCAGUUCAUGUAAACAUCCUACACUCAGCUGUAAUACA(SEQ ID NO: 7) UGGAUUGGCUGGGAGGUGGAUGUUUACUUCAGCUGACUUGGA (SEQ ID NO: 8)hsa-miR-21 uagcuuaucagacugauguugaUGUCGGGUAGCUUAUCAGACUGAUGUUGACUGUUGAAUCUCAUGGC (SEQ ID NO: 9)AACACCAGUCGAUGGGCUGUCUGACA (SEQ ID NO: 10) hsa-miR-142-5pcauaaaguagaaagcacuacu GACAGUGCAGUCACCCAUAAAGUAGAAAGCACUACUAACAGCACUG(SEQ ID NO: 11) GAGGGUGUAGUGUUUCCUACUUUAUGGAUGAGUGUACUGUG(SEQ ID NO: 12) hsa-miR-27a uucacaguggcuaaguuccgcCUGAGGAGCAGGGCUUAGCUGCUUGUGAGCAGGGUCCACACCAAGU (SEQ ID NO: 265)CGUGUUCACAGUGGCUAAGUUCCGCCCCCCAG (SEQ ID NO: 13) hsa-miR-16uagcagcacguaaauauuggcg GUCAGCAGUGCCUUAGCAGCACGUAAAUAUUGGCGUUAAGAUUCUA(SEQ ID NO: 14) AAUUAUCUCCAGUAUUAACUGUGCUGCUGAAGUAAGGUUGAC(SEQ ID NO: 16) uagcagcacguaaauauuggcgGUUCCACUCUAGCAGCACGUAAAUAUUGGCGUAGUGAAAUAUAUAU (SEQ ID NO: 15)UAAACACCAAUAUUACUGUGCUGCUUUAGUGUGAC (SEQ ID NO: 17) hsa-miR-374auuauaauacaaccugauaagug UACAUCGGCCAUUAUAAUACAACCUGAUAAGUGUUAUAGCACUUAU(SEQ ID NO: 18) CAGAUUGUAUUGUAAUUGUCUGUGUA (SEQ ID NO: 19) hsa-miR-374bauauaauacaaccugcuaagug ACUCGGAUGGAUAUAAUACAACCUGCUAAGUGUCCUAGCACUUAGC(SEQ ID NO: 20) AGGUUGUAUUAUCAUUGUCCGUGUCU (SEQ ID NO: 21) hsa-miR-101uacaguacugugauaacugaa UGCCCUGGCUCAGUUAUCACAGUGCUGAUGCUGUCUAUUCUAAAGG(SEQ ID NO: 22) UACAGUACUGUGAUAACUGAAGGAUGGCA uacaguacugugauaacugaa(SEQ ID NO: 24) (SEQ ID NO: 23)ACUGUCCUUUUUCGGUUAUCAUGGUACCGAUGCUGUAUAUCUGAAAGGUACAGUACUGUGAUAACUGAAGAAUGGUGGU (SEQ ID NO: 25) hsa-miR-340uuauaaagcaaugagacugauu UUGUACCUGGUGUGAUUAUAAAGCAAUGAGACUGAUUGUCAUAUGU(SEQ ID NO: 26) CGUUUGUGGGAUCCGUCUCAGUUACUUUAUAGCCAUACCUGGUAUC UUA(SEQ ID NO: 27) hsa-miR-30e uguaaacauccuugacuggaagGGGCAGUCUUUGCUACUGUAAACAUCCUUGACUGGAAGCUGUAAGG (SEQ ID NO: 28)UGUUCAGAGGAGCUUUCAGUCGGAUGUUUACAGCGGCAGGCUGCCA (SEQ ID NO: 29)hsa-miR-29c uagcaccauuugaaaucgguuaAUCUCUUACACAGGCUGACCGAUUUCUCCUGGUGUUCAGAGUCUGU (SEQ ID NO: 30)UUUUGUCUAGCACCAUUUGAAAUCGGUUAUGAUGUAGGGGGA (SEQ ID NO: 31) hsa-miR-29auagcaccaucugaaaucgguua AUGACUGAUUUCUUUUGGUGUUCAGAGUCAAUAUAAUUUUCUAGCA(SEQ ID NO: 32) CCAUCUGAAAUCGGUUAU (SEQ ID NO: 33) hsa-miR-223ugucaguuugucaaauacccca CCUGGCCUCCUGCAGUGCCACGCUCCGUGUAUUUGACAAGCUGAGU(SEQ ID NO: 34) UGGACACUCCAUGUGGUAGAGUGUCAGUUUGUCAAAUACCCCAAGUGCGGCACAUGCUUACCAG (SEQ ID NO: 35) hsa-miR-26a uucaaguaauccaggauaggcuGUGGCCUCGUUCAAGUAAUCCAGGAUAGGCUGUGCAGGUCCCAAUG (SEQ ID NO: 36)GGCCUAUUCUUGGUUACUUGCACGGGGACGC uucaaguaauccaggauaggcu (SEQ ID NO: 38)(SEQ ID NO: 37) GGCUGUGGCUGGAUUCAAGUAAUCCAGGAUAGGCUGUUUCCAUCUGUGAGGCCUAUUCUUGAUUACUUGUUUCUGGAGGCAGCU (SEQ ID NO: 39) hsa-miR-26buucaaguaauucaggauaggu CCGGGACCCAGUUCAAGUAAUUCAGGAUAGGUUGUGUGCUGUCCAG(SEQ ID NO: 40) CCUGUUCUCCAUUACUUGGCUCGGGGACCGG (SEQ ID NO: 41)hsa-miR-24 uggcucaguucagcaggaacagCUCCGGUGCCUACUGAGCUGAUAUCAGUUCUCAUUUUACACACUGG (SEQ ID NO: 42)CUCAGUUCAGCAGGAACAGGAG uggcucaguucagcaggaacag (SEQ ID NO: 44)(SEQ ID NO: 43) CUCUGCCUCCCGUGCCUACUGAGCUGAAACACAGUUGGUUUGUGUACACUGGCUCAGUUCAGCAGGAACAGGG (SEQ ID NO: 45) hsa-miR-181aaacauucaacgcugucggugagu UGAGUUUUGAGGUUGCUUCAGUGAACAUUCAACGCUGUCGGUGAGU(SEQ ID NO: 46) UUGGAAUUAAAAUCAAAACCAUCGACCGUUGAUUGUACCCUAUGGCaacauucaacgcugucggugagu UAACCAUCAUCUACUCCA (SEQ ID NO: 47)(SEQ ID NO: 48) AGAAGGGCUAUCAGGCCAGCCUUCAGAGGACUCCAAGGAACAUUCAACGCUGUCGGUGAGUUUGGGAUUUGAAAAAACCACUGACCGUUGAC UGUACCUUGGGGUCCUUA(SEQ ID NO: 49) hsa-miR-103 agcagcauuguacagggcuaugaUACUGCCCUCGGCUUCUUUACAGUGCUGCCUUGUUGCAUAUGGAUC (SEQ ID NO: 50)AAGCAGCAUUGUACAGGGCUAUGAAGGCAUUG agcagcauuguacagggcuauga (SEQ ID NO: 54)(SEQ ID NO: 51) UUGUGCUUUCAGCUUCUUUACAGUGCUGCCUUGUAGCAUUCAGGUCucauagcccuguacaaugcugcu AAGCAGCAUUGUACAGGGCUAUGAAAGAACCA (SEQ ID NO: 52)(SEQ ID NO: 55) ucauagcccuguacaaugcugcuUCAUAGCCCUGUACAAUGCUGCUUGAUCCAUAUGCAACAAGGCAGC (SEQ ID NO: 53)ACUGUAAAGAAGCCGA (SEQ ID NO: 56)UCAUAGCCCUGUACAAUGCUGCUUGACCUGAAUGCUACAAGGCAGC ACUGUAAAGAAGCUGA(SEQ ID NO: 57) hsa-miR-155 uuaaugcuaaucgugauagggguCUGUUAAUGCUAAUCGUGAUAGGGGUUUUUGCCUCCAACUGACUCC (SEQ ID NO: 58)UACAUAUUAGCAUUAACAG (SEQ ID NO: 59) hsa-miR-532-3pcaugccuugaguguaggaccgu CGACUUGCUUUCUCUCCUCCAUGCCUUGAGUGUAGGACCGUUGGCA(SEQ ID NO: 60) UCUUAAUUACCCUCCCACACCCAAGGCUUGCAAAAAAGCGAGCCU(SEQ ID NO: 61) hsa-miR-320c aaaagcuggguugagaggguUUUGCAUUAAAAAUGAGGCCUUCUCUUCCCAGUUCUUCCCAGAGUC (SEQ ID NO: 62)AGGAAAAGCUGGGUUGAGAGGGUAGAAAAAAAAUGAUGUAGG aaaagcuggguugagagggu(SEQ ID NO: 64) (SEQ ID NO: 63)CUUCUCUUUCCAGUUCUUCCCAGAAUUGGGAAAAGCUGGGUUGAGA GGGU (SEQ ID NO: 65)hsa-miR-27b uucacaguggcuaaguucugcACCUCUCUAACAAGGUGCAGAGCUUAGCUGAUUGGUGAACAGUGAU (SEQ ID NO: 66)UGGUUUCCGCUUUGUUCACAGUGGCUAAGUUCUGCACCUGAAGAGA AGGUG (SEQ ID NO: 67)hsa-miR-664 uauucauuuauccccagccuacaGAACAUUGAAACUGGCUAGGGAAAAUGAUUGGAUAGAAACUAUUAU (SEQ ID NO: 68)UCUAUUCAUUUAUCCCCAGCCUACAAAAUGAAAAAA (SEQ ID NO: 69) hsa-miR-432-5pucuuggaguaggucauugggugg UGACUCCUCCAGGUCUUGGAGUAGGUCAUUGGGUGGAUCCUCUAUU(SEQ ID NO: 70) UCCUUACGUGGGCCACUGGAUGGCUCCUCCAUGUCUUGGAGUAGAU CA(SEQ ID NO: 71) hsa-miR-92a uauugcacuugucccggccuguCUUUCUACACAGGUUGGGAUCGGUUGCAAUGCUGUGUUUCUGUAUG (SEQ ID NO: 72)GUAUUGCACUUGUCCCGGCCUGUUGAGUUUGG uauugcacuugucccggccugu (SEQ ID NO: 74)(SEQ ID NO: 73) UCAUCCCUGGGUGGGGAUUUGUUGCAUUACUUGUGUUCUAUAUAAAGUAUUGCACUUGUCCCGGCCUGUGGAAGA (SEQ ID NO: 75) hsa-miR-99bcacccguagaaccgaccuugcg GGCACCCACCCGUAGAACCGACCUUGCGGGGCCUUCGCCGCACACA(SEQ ID NO: 76) AGCUCGUGUCUGUGGGUCCGUGUC (SEQ ID NO: 77) hsa-miR-146augagaacugaauuccauggguu CCGAUGUGUAUCCUCAGCUUUGAGAACUGAAUUCCAUGGGUUGUGU(SEQ ID NO: 78) CAGUGUCAGACCUCUGAAAUUCAGUUCUUCAGCUGGGAUAUCUCUG UCAUCGU(SEQ ID NO: 79) hsa-miR-150 ucucccaacccuuguaccagugCUCCCCAUGGCCCUGUCUCCCAACCCUUGUACCAGUGCUGGGCUCA (SEQ ID NO: 80)GACCCUGGUACAGGCCUGGGGGACAGGGACCUGGGGAC (SEQ ID NO: 81) hsa-miR-328cuggcccucucugcccuuccgu UGGAGUGGGGGGGCAGGAGGGGCUCAGGGAGAAAGUGCAUACAGCC(SEQ ID NO: 82) CCUGGCCCUCUCUGCCCUUCCGUCCCCUG (SEQ ID NO: 83)hsa-miR-532-3p ccucccacacccaaggcuugcaCGACUUGCUUUCUCUCCUCCAUGCCUUGAGUGUAGGACCGUUGGCA (SEQ ID NO: 84)UCUUAAUUACCCUCCCACACCCAAGGCUUGCAAAAAAGCGAGCCU (SEQ ID NO: 85)hsa-miR-1260 aucccaccucugccaccaACCUUUCCAGCUCAUCCCACCUCUGCCACCAAAACACUCAUCGCGG (SEQ ID NO: 86)GGUCAGAGGGAGUGCCAAAAAAGGUAA (SEQ ID NO: 87) hsa-miR-423ugaggggcagagacgagacuuu AUAAAGGAAGUUAGGCUGAGGGGCAGAGAGCGAGACUUUUCUAUUU(SEQ ID NO: 88) UCCAAAAGCUCGGUCUGAGGCCCCUCAGUCUUGCUUCCUAACCCGC GC(SEQ ID NO: 89) hsa-miR-93 caaagugcuguucgugcagguagGGAGCUUAUCAGAAUCUCCAGGGGUACUUUAUAAUUUCAAAAAGUC (SEQ ID NO: 92)CCCCAGGUGUGAUUCUGAUUUGCUUC (SEQ ID NO: 91) hsa-miR-221agcuacauugucugcuggguuuc CUGGGGGCUCCAAAGUGCUGUUCGUGCAGGUAGUGUGAUUACCCAA(SEQ ID NO: 94) CCUACUGCUGAGCUAGCACUUCCCGAGCCCCCGG (SEQ ID NO: 93)hsa-miR-20a uaaagugcuuauagugcagguagGUAGCACUAAAGUGCUUAUAGUGCAGGUAGUGUUUAGUUAUCUACU (SEQ ID NO: 96)GCAUUAUGAGCACUUAAAGUACUGC (SEQ ID NO: 97) hsa-miR-30cuguaaacauccuacacucucagc ACCAUGCUGUAGUGUGUGUAAACAUCCUACACUCUCAGCUGUGAGC(SEQ ID NO: 98) UCAAGGUGGCUGGGAGAGGGUUGUUUACUCCUUCUGCCAUGGAuguaaacauccuacacucucagc (SEQ ID NO: 100) (SEQ ID NO: 99)AGAUACUGUAAACAUCCUACACUCUCAGCUGUGGAAAGUAAGAAAGCUGGGAGAAGGCUGUUUACUCUUUCU (SEQ ID NO: 101) hsa-miR-15buagcagcacaucaugguuuaca UUGAGGCCUUAAAGUACUGUAGCAGCACAUCAUGGUUUACAUGCUA(SEQ ID NO: 102) CAGUCAAGAUGCGAAUCAUUAUUUGCUGCUCUAGAAAUUUAAGGAA AUUCAU(SEQ ID NO: 103) hsa-let-7g ugagguaguaguuuguacaguuAGGCUGAGGUAGUAGUUUGUACAGUUUGAGGGUCUAUGAUACCACC (SEQ ID NO: 104)CGGUACAGGAGAUAACUGUACAGGCCACUGCCUUGCCA (SEQ ID NO: 105) hsa-let-7bugagguaguagguugugugguu CGGGGUGAGGUAGUAGGUUGUGUGGUUUCAGGGCAGUGAUGUUGCC(SEQ ID NO: 106) CCUCGGAAGAUAACUAUACAACCUACUGCCUUCCCUG (SEQ ID NO: 107)hsa-let-7a ugagguaguagguuguauaguuUGGGAUGAGGUAGUAGGUUGUAUAGUUUUAGGGUCACACCCACCAC (SEQ ID NO: 108)UGGGAGAUAACUAUACAAUCUACUGUCUUUCCUA ugagguaguagguuguauaguu(SEQ ID NO: 111) (SEQ ID NO: 109)AGGUUGAGGUAGUAGGUUGUAUAGUUUAGAAUUACAUCAAGGGAGA ugagguaguagguuguauaguuUAACUGUACAGCCUCCUAGCUUUCCU (SEQ ID NO: 110) (SEQ ID NO: 112)GGGUGAGGUAGUAGGUUGUAUAGUUUGGGGCUCUGCCCUGCUAUGGGAUAACUAUACAAUCUACUGUCUUUCCU (SEQ ID NO: 113) hsa-miR-574-3pugagugugugugugugagugugu GGGACCUGCGUGGGUGCGGGCGUGUGAGUGUGUGUGUGUAGUGUGU(SEQ ID NO: 114) GUCGCUCCGGGUCCACGCUCAUGCACACCCACACGCCCACACUCAG G(SEQ ID NO: 115) hsa-miR-19a ugugcaaaucuaugcaaaacugaGCAGUCCUCUGUUAGUUUUGCAUAGUUGCACUACAAGAAGAAUGUA (SEQ ID NO: 116)GUUGUGCAAAUCUAUGCAAAACUGAUGGUGGCCUGC (SEQ ID NO: 117) hsa-let-7fugagguaguagauuguauaguu UCAGAGUGAGGUAGUAGAUUGUAUAGUUGUGGGGUAGUGAUUUUAC(SEQ ID NO: 118) CCUGUUCAGGAGAUAACUAUACAAUCUAUUGCCUUCCCUGAugagguaguagauuguauaguu (SEQ ID NO: 120) (SEQ ID NO: 119)UGUGGGAUGAGGUAGUAGAUUGUAUAGUUUUAGGGUCAUACCCCAUCUUGGAGAUAACUAUACAGUCUACUGUCUUUCCCACG (SEQ ID NO: 121) hsa-miR-140-5pcagugguuuuacccuaugguag UGUGUCUCUCUCUGUGUCCUGCCAGUGGUUUUACCCUAUGGUAGGU(SEQ ID NO: 122) UACGUCAUGCUGUUCUACCACAGGGUAGAACCACGGACAGGAUACC GGGGCACC(SEQ ID NO: 123) hsa-miR-30a uguaaacauccucgacuggaagGCGACUGUAAACAUCCUCGACUGGAAGCUGUGAAGCCACAGAUGGG (SEQ ID NO: 124)CUUUCAGUCGGAUGUUUGCAGCUGC (SEQ ID NO: 125) hsa-miR-190ugauauguuugauauauuaggu UGCAGGCCUCUGUGUGAUAUGUUUGAUAUAUUAGGUUGUUAUUUAA(SEQ ID NO: 126) UCCAACUAUAUAUCAAACAUAUUCCUACAGUGUCUUGCC(SEQ ID NO: 127) hsa-miR-500 uaauccuugcuaccugggugagaGCUCCCCCUCUCUAAUCCUUGCUACCUGGGUGAGAGUGCUGUCUGA (SEQ ID NO: 128)AUGCAAUGCACCUGGGCAAGGAUUCUGAGAGCGAGAGC aauccuugcuaccugggu(SEQ ID NO: 130) (SEQ ID NO: 129)CCCCCUCUCUAAUCCUUGCUACCUGGGUGAGAGUGCUUUCUGAAUGCAGUGCACCCAGGCAAGGAUUCUGCAAGGGGGA (SEQ ID NO: 131) hsa-let-7iugagguaguaguuugugcuguu CUGGCUGAGGUAGUAGUUUGUGCUGUUGGUCGGGUUGUGACAUUGC(SEQ ID NO: 132) CCGCUGUGGAGAUAACUGCGCAAGCUACUGCCUUGCUA (SEQ ID NO: 133)hsa-miR-23a aucacauugccagggauuuccGGCCGGCUGGGGUUCCUGGGGAUGGGAUUUGCUUCCUGUCACAAAU (SEQ ID NO: 134)CACAUUGCCAGGGAUUUCCAACCGACC (SEQ ID NO: 135) hsa-miR-142-3pcauaaaguagaaagcacuacu GACAGUGCAGUCACCCAUAAAGUAGAAAGCACUACUAACAGCACUG(SEQ ID NO: 136) GAGGGUGUAGUGUUUCCUACUUUAUGGAUGAGUGUACUGUG(SEQ ID NO: 137) hsa-miR-15a uagcagcacauaaugguuugugCCUUGGAGUAAAGUAGCAGCACAUAAUGGUUUGUGGAUUUUGAAAA (SEQ ID NO: 138)GGUGCAGGCCAUAUUGUGCUGCCUCAAAAAUACAAGG (SEQ ID NO: 139) hsa-miR-191caacggaaucccaaaagcagcug CGGCUGGACAGCGGGCAACGGAAUCCCAAAAGCAGCUGUUGUCUCC(SEQ ID NO: 140) AGAGCAUUCCAGCUGCGCUUGGAUUUCGUCCCCUGCUCUCCUGCCU(SEQ ID NO: 141) hsa-miR-720 ucucgcuggggccuccaCCGGAUCUCACACGGUGGUGUUAAUAUCUCGCUGGGGCCUCCAAAA (SEQ ID NO: 142)UGUUGUGCCCAGGGGUGUUAGAGAAAACACCACACUUUGAGAUGAA UUAAGAGUCCUUUAUUAG(SEQ ID NO: 143) hsa-miR-320a aaaagcuggguugagagggcgaGCUUCGCUCCCCUCCGCCUUCUCUUCCCGGUUCUUCCCGGAGUCGG (SEQ ID NO: 144)GAAAAGCUGGGUUGAGAGGGCGAAAAAGGAUGAGGU (SEQ ID NO: 145) hsa-miR-520gacaaagugcuucccuuuagagug UCCCAUGCUGUGACCCUCUAGAGGAAGCACUUUCUGUUUGUUGUCU uGAGAAAAAACAAAGUGCUUCCCUUUAGAGUGUUACCGUUUGGGA (SEQ ID NO: 146)(SEQ ID NO: 147) hsa-miR-204 uucccuuugucauccuaugccuGGCUACAGUCUUUCUUCAUGUGACUCGUGGACUUCCCUUUGUCAUC (SEQ ID NO: 148)CUAUGCCUGAGAAUAUAUGAAGGAGGCUGGGAAGGCAAAGGGACGU UCAAUUGUCAUCACUGGC(SEQ ID NO: 149) hsa-miR-708 aaggagcuuacaaucuagcugggAACUGCCCUCAAGGAGCUUACAAUCUAGCUGGGGGUAAAUGACUUG (SEQ ID NO: 252)CACAUGAACACAACUAGACUGUGAGCUUCUAGAGGGCAGGGA (SEQ ID NO: 253) hsa-miR-197uucaccaccuucuccacccagc GGCUGUGCCGGGUAGAGAGGGCAGUGGGAGGUAAGAGCUCUUCACC(SEQ ID NO: 254) CUUCACCACCUUCUCCACCCAGCAUGGCC (SEQ ID NO: 255)hsa-miR-1274a GUCCCUGUUCAGGCGCCA (SEQ ID NO: 256) hsa-miR-1274bUCCCUGUUCGGGCGCCA (SEQ ID NO: 264)

MicroRNA markers decreased in CD14⁺CD16⁻ or CD14⁺CD16⁺ monocytes insubjects having a neurodegenerative disorder relative to healthycontrols (CD14⁺CD16⁻ or CD14⁺CD16⁻ monocytes in healthy controls) arelisted in Table 2.

TABLE 2List of microRNAs decreased in CD14⁺CD16⁻ or CD14⁺CD16⁻ monocytes fromsubjects having a neurodegenerative disease compared to healthy controlsmiRNA Mature miRNA sequence Precursor miRNA sequence hsa-miR-518fgaaagcgcuucucuuuagagg UCUCAUGCUGUGACCCUCUAGAGGGAAGCACUUUCUCUUGUCUAAA(SEQ ID NO: 150) AGAAAAGAAAGCGCUUCUCUUUAGAGGAUUACUCUUUGAGA(SEQ ID NO: 151) hsa-miR-206 uggaauguaaggaaguguguggUGCUUCCCGAGGCCACAUGCUUCUUUAUAUCCCCAUAUGGAUUACU (SEQ ID NO: 152)UUGCUAUGGAAUGUAAGGAAGUGUGUGGUUUCGGCAAGUG (SEQ ID NO: 153) hsa-miR-204uucccuuugucauccauaugcc GGCUACAGUCUUUCUUCAUGUGACUCGUGGACUUCCCUUUGUCAUC uCUAUGCCUGAGAAUAUAUGAAGGAGGCUGGGAAGGCAAAGGGACGU (SEQ ID NO: 154)UCAAUUGUCAUCACUGGC (SEQ ID NO: 155) hsa-miR-137 uuauugcuuaagaauacgcguaGGUCCUCUGACUCUCUUCGGUGACGGGUAUUCUUGGGUGGAUAAUA gCGGAUUACGUUGUUAUUGCUUAAGAAUACGCGUAGUCGAGGAGAGU (SEQ ID NO: 156)ACCAGCGGCA (SEQ ID NO: 157) hsa-miR-453 AGGUUGUCCGUGGUGAGUUCGCUGGUACUCGGAGGGAGGUUGUCCGUGGUGAGUUCGCAUUAUUUAAU AGAUGCCCAAUACACGGUCGACCUCUUUUCGGUAUCA (SEQ ID NO: 257) (SEQ ID NO: 258)hsa-miR-603 cacacaugcaauuacuuuugcGAUUGAUGCUGUUGGUUUGGUGCAAAAGUAAUUGCAGUGCUUCCCA (SEQ ID NO: 158)UUUAAAAGUAAUGGCACACACUGCAAUUACUUUUGCUUCCAACUUA AUACUU (SEQ ID NO: 159)hsa-miR-1297 uucaaguaauucaggugUGUUUAUCUCUAGGGUUGAUCUAUUAGAAUUACUUAUCUGAGCCAA (SEQ ID NO: 160)AGUAAUUCAAGUAAUUCAGGUGUAGUGAAAC (SEQ ID NO: 161) hsa-miR-192cugaccuaugaauugacagcc GCCGAGACCGAGUGCACAGGGCUCUGACCUAUGAAUUGACAGCCAG(SEQ ID NO: 162) UGCUCUCGUCUCCCCUCUGGCUGCCAAUUCCAUAGGUCACAGGUAUGUUCGCCUCAAUGCCAGCC (SEQ ID NO: 163) hsa-miR-526a cucuagagggaagcacuuucugCUCAGGCUGUGACCCUCUAGAGGGAAGCACUUUCUGUUGCUUGAAA (SEQ ID NO: 164)GAAGAGAAAGCGCUUCCUUUUAGAGGAUUACUCUUUGAG cucuagagggaagcacuuucug(SEQ ID NO: 166) (SEQ ID NO: 165)GUGACCCUCUAGAGGGAAGCACUUUCUGUUGAAAGAAAAGAACAUG CAUCCUUUCAGAGGGUUAC(SEQ ID NO: 167) hsa-miR-615-5p ggggguccccggugcucggaucCUCGGGAGGGGCGGGAGGGGGGUCCCCGGUGCUCGGAUCUCGAGGG (SEQ ID NO: 168)UGCUUAUUGUUCGGUCCGAGCCUGGGUCUCCCUCUUCCCCCCAACC CCCC (SEQ ID NO: 169)hsa-miR-655 auaauacaugguuaaccucuuuAACUAUGCAAGGAUAUUUGAGGAGAGGUUAUCCGUGUUAUGUUCGC (SEQ ID NO: 170)UUCAUUCAUCAUGAAUAAUACAUGGUUAACCUCUUUUUGAAUAUCA GACUC (SEQ ID NO: 171)hsa-miR-450b-5p uuuugcaauauguuccugaauaGCAGAAUUAUUUUUGCAAUAUGUUCCUGAAUAUGUAAUAUAAGUGU (SEQ ID NO: 172)AUUGGGAUCAUUUUGCAUCCAUAGUUUUGUAU (SEQ ID NO: 173) hsa-miR-548b-3paaaaguaauugugguuuuggcc CAGACUAUAUAUUUAGGUUGGCGCAAAAGUAAUUGUGGUUUUGGCC(SEQ ID NO: 174) UUUAUUUUCAAUGGCAAGAACCUCAGUUGCUUUUGUGCCAACCUAA UACUU(SEQ ID NO: 175) hsa-miR-584 uuaugguuugccugggacugagUAGGGUGACCAGCCAUUAUGGUUUGCCUGGGACUGAGGAAUUUGCU (SEQ ID NO: 176)GGGAUAUGUCAGUUCCAGGCCAACCAGGCUGGUUGGUCUCCCUGAA GCAAC (SEQ ID NO: 177)hsa-miR-548f aaaaacuguaauuacuuuuAUUAGGUUGGUGCAAAAGUAAUCACAGUUUUUGACAUUACUUUCAA (SEQ ID NO: 178)AGACAAAAACUGUAAUUACUUUUGGACCAACCUAAUAG aaaaacuguaauuacuuuu(SEQ ID NO: 183) (SEQ ID NO: 179)UAAUAACUAUUAGGUUGGUGCGAACAUAAUUGCAGUUUUUAUCAUU aaaaacuguaauuacuuuuACUUUUAAUGGCAAAAACUGUAAUUACUUUUGCACCAACCUAAUAU (SEQ ID NO: 180) UUUAGUaaaaacuguaauuacuuuu (SEQ ID NO: 184) (SEQ ID NO: 181)AUUAGGUUGGUGCAAACCUAAUUGCAAUUUUUGCAGUUUUUUUAAG aaaaacuguaauuacuuuuUAAUUGCAAAAACUGUAAUUACUUUUGCACCAACCUAAUAC (SEQ ID NO: 182)(SEQ ID NO: 185) GAGUUCUAACGUAUUAGGUUGGUGCAAAAGUAAUAGUGGUUUUUGCCAUUAAAAGUAAUGACAAAAACUGUAAUUACUUUUGGAACAAUAUU AAUAGAAUUUCAG(SEQ ID NO: 186) UAUUAGGUUGCUGCAAAAGUAAUCAUGUUUUUUUCCAUUGUAAGUAAUGGGAAAAACUGUAAUUACUUUUGUACCAACCUAAUAGC (SEQ ID NO: 187) hsa-miR-300uauacaagggcagacucucucu UGCUACUUGAAGAGAGGUAAUCCUUCACGCAUUUGCUUUACUUGCA(SEQ ID NO: 188) AUGAUUAUACAAGGGCAGACUCUCUCUGGGGAGCAAA (SEQ ID NO: 189)hsa-miR-302c uaagugcuuccauguuucagugCCUUUGCUUUAACAUGGGGGUACCUGCUGUGUGAAACAAAAGUAAG g UGCUUCCAUGUUUCAGUGGAGG(SEQ ID NO: 190) (SEQ ID NO: 191) hsa-miR-328 cuggcccucucugcccuuccguUGGAGUGGGGGGGCAGGAGGGGCUCAGGGAGAAAGUGCAUACAGCC (SEQ ID NO: 82)CCUGGCCCUCUCUGCCCUUCCGUCCCCUG (SEQ ID NO: 83) hsa-miR-421aucaacagacauuaauugggcg GCACAUUGUAGGCCUCAUUAAAUGUUUGUUGAAUGAAAAAAUGAAU cCAUCAACAGACAUUAAUUGGGCGCCUGCUCUGUGAUCUC (SEQ ID NO: 192)(SEQ ID NO: 193) hsa-miR-580 uugagaaugaugaaucauuaggAUAAAAUUUCCAAUUGGAACCUAAUGAUUCAUCAGACUCAGAUAUU (SEQ ID NO: 194)UAAGUUAACAGUAUUUGAGAAUGAUGAAUCAUUAGGUUCCGGUCAG AAAUU (SEQ ID NO: 195)hsa-miR-651 uuuaggauaagcuugacuuuugAAUCUAUCACUGCUUUUUAGGAUAAGCUUGACUUUUGUUCAAAUAA (SEQ ID NO: 196)AAAUGCAAAAGGAAAGUGUAUCCUAAAAGGCAAUGACAGUUUAAUG UGUUU (SEQ ID NO: 197)hsa-miR-379 ugguagacuauggaacguaggAGAGAUGGUAGACUAUGGAACGUAGGCGUUAUGAUUUCUGACCUAU (SEQ ID NO: 198)GUAACAUGGUCCACUAACUCU (SEQ ID NO: 199) hsa-miR-193a-3pugggucuuugcgggcgagauga CGAGGAUGGGAGCUGAGGGCUGGGUCUUUGCGGGCGAGAUGAGGGU(SEQ ID NO: 200) GUCGGAUCAACUGGCCUACAAAGUCCCAGUUCUCGGCCCCCG(SEQ ID NO: 201) hsa-miR-515-3p uucuccaaaagaaagcacuuucUCUCAUGCAGUCAUUCUCCAAAAGAAAGCACUUUCUGUUGUCUGAA ugAGCAGAGUGCCUUCUUUUGGAGCGUUACUGUUUGAGA (SEQ ID NO: 202) (SEQ ID NO: 204)uucuccaaaagaaagcacuuuc UCUCAUGCAGUCAUUCUCCAAAAGAAAGCACUUUCUGUUGUCUGAA ugAGCAGAGUGCCUUCUUUUGGAGCGUUACUGUUUGAGA (SEQ ID NO: 203) (SEQ ID NO: 205)hsa-miR-598 uacgucaucguugucaucgucaGCUUGAUGAUGCUGCUGAUGCUGGCGGUGAUCCCGAUGGUGUGAGC (SEQ ID NO: 206)UGGAAAUGGGGUGCUACGUCAUCGUUGUCAUCGUCAUCAUCAUCAU CCGAG (SEQ ID NO: 207)hsa-miR-513a-5p uucacagggaggugucauGGGAUGCCACAUUCAGCCAUUCAGCGUACAGUGCCUUUCACAGGGA (SEQ ID NO: 208)GGUGUGCAUUUAUGUGAACUAAAAUAUAAAUUUCACCUUUCUGAGA uucacagggaggugucauAGGGUAAUGUACAGCAUGCACUGCAUAUGUGGUGUCCC (SEQ ID NO: 209) (SEQ ID NO: 210)GGAUGCCACAUUCAGCCAUUCAGUGUGCAGUGCCUUUCACAGGGAGGUGUCAUUUAUGUGAACUAAAAUAUAAAUUUCACCUUUCUGAGAAGGGUAAUGUACAGCAUGCACUGCAUAUGUGGUGUCC (SEQ ID NO: 211) hsa-miR-640augauccaggaaccugccucu GUGACCCUGGGCAAGUUCCUGAAGAUCAGACACAUCAGAUCCCUUA(SEQ ID NO: 212) UCUGUAAAAUGGGCAUGAUCCAGGAACCUGCCUCUACGGUUGCCUU GGGG(SEQ ID NO: 213) hsa-miR-548g aaaacuguaauuacuuuuguacAGUUAUUAGAUUAGUGCAAAAGUAAUUGCAGUUUUUGCAUUACGUU (SEQ ID NO: 214)CUAUGGCAAAACUGUAAUUACUUUUGUACCAACAUAAUACUUC (SEQ ID NO: 215)hsa-miR-1206 uguucauguagauguuuaagcCAGUGUUCAUGUAGAUGUUUAAGCUCUUGCAGUAGGUUUUUGCAAG (SEQ ID NO: 216)CUAGUGAACGCUG (SEQ ID NO: 217) hsa-miR-383 agaucagaaggugauuguggcuCUCCUCAGAUCAGAAGGUGAUUGUGGCUUUGGGUGGAUAUUAAUCA (SEQ ID NO: 218)GCCACAGCACUGCCUGGUCAGAAAGAG (SEQ ID NO: 219) hsa-miR-649aaaccuguguuguucaagaguc GGCCUAGCCAAAUACUGUAUUUUUGAUCGACAUUUGGUUGAAAAAU(SEQ ID NO: 220) AUCUAUGUAUUAGUAAACCUGUGUUGUUCAAGAGUCCACUGUGUUU UGCUG(SEQ ID NO: 221) hsa-miR-592 uugugucaauaugcgaugauguUAUUAUGCCAUGACAUUGUGUCAAUAUGCGAUGAUGUGUUGUGAUG (SEQ ID NO: 222)GCACAGCGUCAUCACGUGGUGACGCAACAUCAUGACGUAAGACGU CACAAC (SEQ ID NO: 223)hsa-miR-2054 cuguaauauaaauuuaauuuauCUGUAAUAUAAAUUUAAUUUAUUCUCUAUCAUUAAAAAAUGUAUUA u CAG (SEQ ID NO: 224)(SEQ ID NO: 225) hsa-miR-450a uuuugcgauguguuccuaauauAAACGAUACUAAACUGUUUUUGCGAUGUGUUCCUAAUAUGCACUAU (SEQ ID NO: 226)AAAUAUAUUGGGAACAUUUUGCAUGUAUAGUUUUGUAUCAAUAUA uuuugcgauguguuccuaauau(SEQ ID NO: 228) (SEQ ID NO: 227)CCAAAGAAAGAUGCUAAACUAUUUUUGCGAUGUGUUCCUAAUAUGUAAUAUAAAUGUAUUGGGGACAUUUUGCAUUCAUAGUUUUGUAUCAA UAAUAUGG (SEQ ID NO: 229)hsa-miR-362-3p aauccuuggaaccuaggugugaCUUGAAUCCUUGGAACCUAGGUGUGAGUGCUAUUUCAGUGCAACAC gu ACCUAUUCAAGGAUUCAAA(SEQ ID NO: 230) (SEQ ID NO: 231) hsa-miR-193a-3p ugggucuuugcgggcgagaugaCGAGGAUGGGAGCUGAGGGCUGGGUCUUUGCGGGCGAGAUGAGGGU (SEQ ID NO: 232)GUCGGAUCAACUGGCCUACAAAGUCCCAGUUCUCGGCCCCCG (SEQ ID NO: 233) hsa-miR-566gggcgccugugaucccaac GCUAGGCGUGGUGGCGGGCGCCUGUGAUCCCAACUACUCAGGAGGC(SEQ ID NO: 234) UGGGGCAGCAGAAUCGCUUGAACCCGGGAGGCGAAGGUUGCAGUGA GC(SEQ ID NO: 235) hsa-miR-142-3p cauaaaguagaaagcacuacuGACAGUGCAGUCACCCAUAAAGUAGAAAGCACUACUAACAGCACUG (SEQ ID NO: 236)GAGGGUGUAGUGUUUCCUACUUUAUGGAUGAGUGUACUGUG (SEQ ID NO: 237) hsa-miR-15auagcagcacauaaugguuugug CCUUGGAGUAAAGUAGCAGCACAUAAUGGUUUGUGGAUUUUAAAAG(SEQ ID NO: 238) GUGCAGGCCAUAUUGUGCUGCCUCAAAAAUACAAGG (SEQ ID NO: 239)hsa-miR-1537 aaaaccgucuaguuacaguuguACAGCUGUAAUUAGUCAGUUUUCUGUCCUGUCCACACAGAAAACCG (SEQ ID NO: 240)UCUAGUUACAGUUGU (SEQ ID NO: 241) hsa-miR-148b ucagugcaucacagaacuuuguCAAGCACGAUUAGCAUUUGAGGUGAAGUUCUGUUAUACACUCAGGC (SEQ ID NO: 242)UGUGGCUCUCUGAAAGUCAGUGCAUCACAGAACUUUGUCUCGAAAG CUUUCUA (SEQ ID NO: 243)hsa-miR-494 ugaaacauacacgggaaaccucGAUACUCGAAGGAGAGGUUGUCCGUGUUGUCUUCUCUUUAUUUAUG (SEQ ID NO: 244)AUGAAACAUACACGGGAAACCUCUUUUUUAGUAUC (SEQ ID NO: 245) hsa-miR-369-3pagaucgaccguguuauauucgc UUGAAGGGAGAUCGACCGUGUUAUAUUCGCUUUAUUGACUUCGAAU(SEQ ID NO: 246) AAUACAUGGUUGAUCUUUUCUCAG (SEQ ID NO: 247) hsa-miR-10auacccuguagauccgaauuugu GAUCUGUCUGUCUUCUGUAUAUACCCUGUAGAUCCGAAUUUGUGUA gAGGAAUUUUGUGGUCACAAAUUCGUAUCUAGGGGAAUAUGUAGUUG (SEQ ID NO: 248)ACAUAAACACUCCGCUCU (SEQ ID NO: 249) hsa-miR-30d uguaaacauccccgacuggaagGUUGUUGUAAACAUCCCCGACUGGAAGCUGUAAGACACAGCUAAGC (SEQ ID NO: 250)UUUCAGUCAGAUGUUUGCUGCUAC (SEQ ID NO: 251) hsa-miR-660uacccauugcauaucggaguug CUGCUCCUUCUCCCAUACCCAUUGCAUAUCGGAGUUGUAGAAUUCU(SEQ ID NO: 260) CAAAACACCUCCUGUGUGCAUGGAUUACAGGAGGGUGAGCCUUGUCaccuccugugugcauggauua AUCGUG (SEQ ID NO: 261) (SEQ ID NO: 259)

MicroRNA markers increased in monocytes (CD14⁺CD16⁻ or CD14⁺CD16⁺monocytes) or the CSF in subjects having ALS relative to healthycontrols (CD14⁺CD16⁻ or CD14⁺CD16⁻ monocytes, or the CSF in healthycontrols) are listed in Table 3.

TABLE 3 List of microRNAs increased in CD14⁺CD16⁻ or CD14⁺CD16⁺monocytes, or in the CSF in subjects having ALS relative to healthycontrols. hsa-miR-19b hsa-miR-26b hsa-let-7a hsa-miR-106b hsa-miR-24hsa-miR-574-3p hsa-miR-30b hsa-miR-181a hsa-miR-19a hsa-miR-21hsa-miR-103 hsa-let-7f hsa-miR-142-5p hsa-miR-155 hsa-miR-140-5phsa-miR-27a hsa-miR-532-3p hsa-miR-30a hsa-miR-16 hsa-miR-1260hsa-miR-190 hsa-miR-374a hsa-miR-423 hsa-miR-500 hsa-miR-374bhsa-miR-361-5p hsa-let-7i hsa-miR-101 hsa-miR-93 hsa-miR-23a hsa-miR-340hsa-miR-221 hsa-miR-142-3p hsa-miR-30e hsa-miR-20a hsa-miR-15ahsa-miR-29c hsa-miR-30c hsa-let-7b hsa-miR-29a hsa-miR-15b hsa-miR-26ahsa-miR-223 hsa-let-7g

MicroRNA markers decreased in monocytes (CD14⁺CD16⁻ or CD14⁺CD16⁺monocytes) in subjects having ALS relative to healthy controls(CD14⁺CD16⁻ or CD14⁺CD16⁺ monocytes in healthy controls) are listed inTable 4.

TABLE 4 List of microRNAs decreased in CD14⁺CD16⁻ or CD14⁺CD16⁺monocytes in subjects having ALS compared to healthy controls.hsa-miR-518f hsa-miR-655 hsa-miR-421 hsa-miR-383 hsa-miR-206hsa-miR-450b-5p hsa-miR-651 hsa-miR-649 hsa-miR-204 hsa-miR-548b-3phsa-miR-379 hsa-miR-592 hsa-miR-137 hsa-miR-584 hsa-miR-193a-3phsa-miR-2054 hsa-miR-453 hsa-miR-548f hsa-miR-515-3p hsa-miR-566hsa-miR-603 hsa-miR-300 hsa-miR-598 hsa-miR-494 hsa-miR-1297hsa-miR-302c hsa-miR-513a-5p hsa-miR-142-3p hsa-miR-192 hsa-miR-328hsa-miR-640 hsa-miR-1206 hsa-miR-526a hsa-miR-421 hsa-miR-548ghsa-miR-580 hsa-miR-615-5p hsa-miR-660

MicroRNA markers increased in CD14⁺CD16⁻ monocytes from ALS patientsrelative to CD14⁺CD16⁻ monocytes from healthy controls are listed inTable 5.

TABLE 5 List of microRNAs increased in CD14⁺CD16⁻ monocytes from ALSpatients relative to CD14⁺CD16⁻ monocytes from healthy controls.hsa-miR-1260 hsa-let-7g hsa-miR-26a hsa-miR-500 hsa-miR-30a hsa-let-7bhsa-miR-16 hsa-miR-150 hsa-miR-423 hsa-let-7a hsa-miR-374b hsa-miR-30ehsa-miR-361-5p hsa-miR-574-3p hsa-miR-140-5p hsa-miR-29c hsa-miR-93hsa-miR-26b hsa-miR-101 hsa-miR-29a hsa-miR-103 hsa-miR-532-3phsa-miR-142-5p hsa-miR-223 hsa-miR-24 hsa-miR-19a hsa-miR-374ahsa-mIR-423 hsa-miR-221 hsa-let-7f hsa-miR-340 hsa-miR-1260 hsa-miR-20ahsa-miR-27a hsa-miR-21 hsa-miR-30a hsa-miR-30c hsa-miR-106b hsa-miR-155hsa-miR-30b hsa-miR-181a hsa-miR-19b hsa-miR-146a hsa-miR-190hsa-miR-15b

MicroRNAs that are decreased in CD14⁺CD16⁻ monocytes from ALS patientsrelative to CD14⁺CD16⁻ monocytes from healthy controls are listed inTable 6.

TABLE 6 List of microRNAs decreased in CD14⁺CD16⁻ monocytes from ALSpatients relative to CD14⁺CD16⁻ monocytes from healthy controls.hsa-miR-328 hsa-miR-513a-5p hsa-miR-302c hsa-miR-453 hsa-miR-651hsa-miR-640 hsa-miR-2054 hsa-miR-204 hsa-miR-379 hsa-miR-548ghsa-miR-584 hsa-miR-518f hsa-miR-300 hsa-miR-1206 hsa-miR-655hsa-miR-206 hsa-miR-548f hsa-miR-450b-5p hsa-miR-421 hsa-miR-192hsa-miR-193a-3p hsa-miR-548b-3p hsa-miR-615-5p hsa-miR-566 hsa-miR-137hsa-miR-383 hsa-miR-526a hsa-miR-598 hsa-miR-580 hsa-miR-649 hsa-miR-603hsa-miR-515-3p hsa-miR-592 hsa-miR-1297 hsa-miR-660

MicroRNA markers uniquely increased in CD14⁺CD16⁻ monocytes from ALSpatients relative to CD14⁺CD16⁻ monocytes from both MS subjects andhealthy controls are listed in Table 7.

TABLE 7 List of microRNAs uniquely increased in CD14⁺CD16⁻ monocytesfrom ALS patients relative to CD14⁺CD16⁻ monocytes from both MS subjectsand healthy controls hsa-miR-19b hsa-miR-16 hsa-miR-29c hsa-miR-181ahsa-miR-106b hsa-miR-374a hsa-miR-29a hsa-miR-103 hsa-miR-30bhsa-miR-374b hsa-miR-223 hsa-miR-155 hsa-miR-21 hsa-miR-101 hsa-miR-26ahsa-miR-532-3p hsa-miR-142-5p hsa-miR-340 hsa-miR-26b hsa-miR-24hsa-miR-27a hsa-miR-30e

MicroRNA markers uniquely decreased in CD14⁺CD16⁻ monocytes from ALSpatients relative to CD14⁺CD16⁻ monocytes from both MS subjects andhealthy controls are listed in Table 8.

TABLE 8 List of microRNAs uniquely decreased in CD14⁺CD16⁻ monocytesfrom ALS patients relative to CD14⁺CD16⁻ monocytes from both MS subjectsand healthy controls hsa-miR-518f hsa-miR-603 hsa-miR-655 hsa-miR-300hsa-miR-206 hsa-miR-1297 hsa-miR-450b-5p hsa-miR-302c hsa-miR-204hsa-miR-192 hsa-miR-548b-3p hsa-miR-328 hsa-miR-137 hsa-miR-526ahsa-miR-584 hsa-miR-421 hsa-miR-453 hsa-miR-615-5p hsa-miR-548fhsa-miR-580

MicroRNA markers increased in CD14⁺CD16⁺ monocytes from ALS patientsrelative to CD14⁺CD16⁺ monocytes from healthy controls are listed inTable 9.

TABLE 9 List of microRNAs increased in CD14⁺CD16⁺ monocytes from ALSpatients relative to CD14⁺CD16⁺ monocytes from healthy controlshsa-miR-708 hsa-miR-24 hsa-miR-26a hsa-miR-21 hsa-miR-142-5p hsa-miR-103hsa-miR-30b hsa-miR-142-3p hsa-miR-15b hsa-miR-23a hsa-miR-16hsa-miR-340 hsa-miR-223 hsa-miR-29a hsa-miR-15a hsa-let-7i

MicroRNA markers decreased in CD14⁺CD16⁺ monocytes from ALS patientsrelative to CD14⁺CD16⁺ monocytes from healthy controls are listed inTable 10.

TABLE 10 List of microRNAs decreased in CD14⁺CD16⁺ monocytes from ALSpatients relative to CD14⁺CD16⁺ monocytes from healthy controlshsa-miR-598 hsa-miR-494 hsa-miR-142-3p

MicroRNA markers uniquely increased in CSF from subjects having sporadicALS or familial ALS compared to CSF from healthy controls are listed inTable 11.

TABLE 11 List of microRNAs uniquely increased in CSF from subjectshaving sporadic ALS or familial ALS compared to CSF from healthycontrols miRNA Form of ALS hsa-miR-27b Familial and sporadic ALShsa-miR-99b Sporadic ALS hsa-miR-146a Sporadic ALS hsa-miR-150 SporadicALS hsa-miR-328 Familial and Sporadic ALS hsa-miR-532-3p Familial andSporadic ALS

MicroRNA markers increased in monocytes (CD14⁺CD16⁻ or CD14⁺CD16⁺monocytes) in subjects having MS relative to healthy controls(CD14⁺CD16⁻ or CD14⁺CD16⁺ in healthy controls) are listed in Table 12.

TABLE 12 List of microRNAs increased in CD14⁺CD16⁻ or CD14⁺CD16⁺monocytes in subjects having MS relative to CD14⁺CD16⁻ or CD14⁺CD16⁺monocytes in healthy controls. hsa-miR-320c hsa-miR-1260 hsa-miR-19bhsa-miR-340 hsa-let-320a hsa-miR-27b hsa-miR-720 hsa-miR-106bhsa-miR-26b hsa-miR-520g hsa-miR-664 hsa-miR-1274a hsa-let-7ghsa-miR-1260 hsa-miR-204 hsa-miR-432-5p hsa-miR-423 hsa-miR-181ahsa-miR-361-5p hsa-miR-29a hsa-miR-92a hsa-miR-197 hsa-miR-140-5phsa-miR-374b hsa-miR-23a hsa-miR-24 hsa-miR-30a hsa-miR-142-5phsa-let-7a hsa-miR-142-3p hsa-miR-93 hsa-miR-221 hsa-miR-19ahsa-miR-532-3p hsa-miR-103 hsa-miR-20a hsa-miR-361-5p hsa-let-7bhsa-miR-155 hsa-miR-15a hsa-miR-let-7a hsa-miR-103 hsa-miR-221hsa-miR-27a hsa-miR-21 hsa-miR-30c hsa-miR-16 hsa-miR-15b hsa-miR-146ahsa-miR-223 hsa-miR-181a hsa-miR-30b hsa-miR-574-3p hsa-let-7ihsa-miR-1274b hsa-miR-423 hsa-miR-26a hsa-let-7f hsa-let-191

MicroRNA markers decreased in monocytes (CD14⁺CD16⁻ or CD14⁺CD16⁺monocytes) in subjects having MS relative to healthy controls(CD14⁺CD16⁻ or CD14⁺CD16⁺ monocytes in healthy controls) are listed inTable 13.

TABLE 13 List of microRNAs decreased in CD14⁺CD16⁻ or CD14⁺CD16⁺monocytes in subjects having MS compared to CD14⁺CD16⁻ or CD14⁺CD16⁺monocytes in healthy controls hsa-miR-649 hsa-miR-362-3p hsa-miR-603hsa-miR-15a hsa-miR-383 hsa-miR-450b-5p hsa-miR-584 hsa-miR-1537hsa-miR-1206 hsa-miR-302c hsa-miR-204 hsa-miR-148b hsa-miR-548ghsa-miR-548f hsa-miR-526a hsa-miR-369-3p hsa-miR-640 hsa-miR-328hsa-miR-453 hsa-miR-615-5p hsa-miR-592 hsa-miR-580 hsa-miR-2054hsa-miR-10a hsa-miR-598 hsa-miR-421 hsa-miR-655 hsa-miR-30dhsa-miR-515-3p hsa-miR-1297 hsa-miR-518f hsa-miR-494 hsa-miR-513a-5phsa-miR-548b-3p hsa-miR-206 hsa-miR-142-3p hsa-miR-651 hsa-miR-615-5phsa-miR-192 hsa-miR-651 hsa-miR-379 hsa-miR-137 hsa-miR-450ahsa-miR-193a-3p hsa-miR-300 hsa-miR-566

MicroRNA markers increased in CD14⁺CD16⁻ monocytes from MS patientsrelative to CD14⁺CD16⁻ monocytes from MS patients are listed in Table14.

TABLE 14 List of microRNAs increased in CD14⁺CD16⁻ monocytes from MSpatients relative to CD14⁺CD16⁻ monocytes from healthy controls.hsa-miR-720 hsa-miR-20a hsa-let-7g hsa-miR-26b hsa-miR-1274a hsa-miR-93hsa-miR-181a hsa-miR-21 hsa-miR-320c hsa-miR-361-5p hsa-140-5phsa-miR-374b hsa-miR-27b hsa-miR-423 hsa-142-5p hsa-let-7a hsa-miR-664hsa-miR-24 hsa-miR-19a hsa-miR-532-3p hsa-miR-1260 hsa-miR-103hsa-let-7b hsa-miR-155 hsa-miR-423 hsa-miR-16 hsa-miR-15b hsa-miR-27ahsa-miR-197 hsa-miR-30b hsa-miR-574-3p hsa-miR-146a hsa-miR-30ahsa-miR-26a hsa-let-7f hsa-miR-92a hsa-miR-30c hsa-miR-19b hsa-miR-340hsa-miR-1274b hsa-miR-221 hsa-miR-106b hsa-miR-101

MicroRNA markers decreased in CD14⁺CD16⁻ monocytes from MS patientsrelative to CD14⁺CD16⁻ monocytes from healthy patients are listed inTable 15.

TABLE 15 List of microRNAs decreased in CD14⁺CD16⁻ monocytes from MSpatients relative to CD14⁺CD16⁻ monocytes from healthy controls.hsa-miR-649 hsa-miR-193a-3p hsa-miR- hsa-miR-206 615-5p hsa-miR-383hsa-miR-450a hsa-miR-137 hsa-miR-192 hsa-miR-1206 hsa-miR-362-3phsa-miR-300 hsa-miR-566 hsa-miR-548g hsa-miR-450b-5p hsa-miR-603hsa-miR-142-3p hsa-miR-640 hsa-miR-302c hsa-miR-584 hsa-miR-15ahsa-miR-592 hsa-miR-548f hsa-miR-204 hsa-miR-1537 hsa-miR-598hsa-miR-328 hsa-miR-526a hsa-miR-148b hsa-miR-515-3p hsa-miR-580hsa-miR-453 hsa-miR-379 hsa-miR-513a-5p hsa-miR-421 hsa-miR-2054hsa-miR-548b-3p hsa-miR-651 hsa-miR-1297 hsa-miR-655 hsa-miR-518f

MicroRNA markers uniquely increased in CD14⁺CD16⁻ monocytes from MSpatients relative to CD14⁺CD16⁻ monocytes from both ALS subjects andhealthy controls are listed in Table 16.

TABLE 16 List of microRNAs uniquely increased in CD14⁺CD16⁻ monocytesfrom MS patients relative to CD14⁺CD16⁻ monocytes from both ALS subjectsand healthy controls hsa-miR-320c hsa-miR-664 hsa-miR-92a hsa-miR-27bhsa-miR-432-5p

MicroRNA markers uniquely decreased in CD14⁺CD16⁻ monocytes from MSpatients relative to CD14⁺CD16⁻ monocytes from both ALS subjects andhealthy controls are listed in Table 17.

TABLE 17 List of microRNAs uniquely decreased in CD14⁺CD16⁻ monocytesfrom MS patients relative to CD14⁺CD16⁻ monocytes from both ALS subjectsand healthy controls hsa-miR-142-3p hsa-miR-1537 hsa-miR-148bhsa-miR-15a hsa-miR-362-3p

MicroRNA markers increased in CD14⁺CD16⁺ monocytes from MS patientscompared to CD14⁺CD16⁺ monocytes from healthy controls are shown inTable 18.

TABLE 18 List of microRNAs increased in CD14⁺CD16⁺ monocytes from MSpatients compared to CD14+CD16+ monocytes from healthy controlshsa-let-7i hsa-miR-520g hsa-miR-24 hsa-miR-21 hsa-miR-191 hsa-miR-204hsa-miR-30b hsa-miR-16 hsa-miR-1260 hsa-miR-340 hsa-miR-142-3phsa-miR-142-5p hsa-miR-720 hsa-miR-15b hsa-miR-103 hsa-miR-223hsa-miR-1274a hsa-miR-29a hsa-miR-15a hsa-miR-320a hsa-miR-23ahsa-miR-26a

MicroRNA markers decreased in CD14⁺CD16⁺ monocytes from MS patientsrelative to CD14⁺CD16⁺ monocytes from healthy controls are listed inTable 19.

TABLE 19 List of microRNAs decreased in CD14⁺CD16⁺ monocytes from MSpatients relative to CD14⁺CD16⁺ monocytes from healthy controlshsa-miR-369-3p hsa-miR-10a hsa-miR-598 hsa-miR-615-5p hsa-miR-30dhsa-miR-494

Inflammatory markers decreased in CD14⁺CD16⁻ monocytes from patientshaving neurodegenerative disorders relative to CD14⁺CD16⁻ monocytes fromhealthy controls are listed in Table 20.

TABLE 20 List of inflammatory markers decreased in CD14⁺CD16⁻ monocytesfrom patients having neurodegenerative disorders relative to CD14⁺CD16⁻monocytes from healthy controls Protein sequence (NCBI Accession No.;mRNA sequence (NCBI Accession No.; Marker Version No.) Version No.) BCL6NP_001128210; NP_001128210.1 NM_001134738; NM_001134738.1 NP_001124317;NP_001124317.1 NM_001130845; NM_001130845.1 IL1RAP NP_001161401;NP_001161401.1 NM_001167929; NM_001167929.1 NP_001161402; NP_001161402.1NM_001167930; NM_001167930.1 NP_001161403; NP_001161403.1 NM_001167931;NM_001167931.1 PLCB1 NP_056007; NP_056007.1 NM_015192; NM_015192.2NP_877398; NP_877398.1 NM_182734; NM_182734.1 MAFK AAC14426; AAC14426.1AF059194; AF059194.1 NFE2L2 NP_006155; NP_006155.2 NM_006164;NM_006164.3 NP_001138884; NP_001138884.1 NM_001145412; NM_001145412.1NP_001138885; NP_001138885.1 NM_001145413; NM_001145413.1 DDIT3NP_001181982; NP_001181982.1 NM_001195053; NM_001195053.1 NP_001181983;NP_001181983.1 NM_001195054; NM_001195054.1 NP_001181984TN;NP_001181984.1 NM_001195055; NM_001195055.1 NP_001181985; NP_001181985.1NM_001195056; NM_001195056.1 NP_004074; NP_004074.2 NM_004083;NM_004083.5 GNAQ NP_002063; NP_002063.2 NM_002072; NM_002072.3 RAPGEF2NP_055062; NP_055062.1 NM_014247; NM_014247.2 MAFG NP_002350;NP_002350.1 NM_002359; NM_002359.3 NP_116100; NP_116100.2 NM_032711;NM_032711.3 PTK2N NP_722560; NP_722560.1 NM_153831; NM_153831.3NP_005598; NP_005598.3 NM_005607; NM_005607.4 NP_001186578;NP_001186578.1 NM_001199649; NM_001199649.1 MKNK1 NP_003675; NP_003675.2NM_003684; NM_003684.4 NP_945324; NP_945324.1 NM_198973; NM_198973.2NP_001129025; NP_001129025.1 NM_001135553; NM_001135553.1 RIPK1NP_003795; NP_003795.2 NM_003804; NM_003804.3 IL15 NP_751915;NP_751915.1 NM_172175; NM_172175.2 NP_000576; NP_000576.1 NM_000585;NM_000585.4 MAP3K1 NP_005912; NP_005912.1 NM_005921; NM_005921.1PPP1R12B NP_001184060; NP_001184060.1 NM_001197131; NM_001197131.1NP_001161330; NP_001161330.1 NM_001167858; NM_001167858.1 NP_001161329;NP_001161329.1 NM_001167857; NM_001167857.1 NP_115287; NP_115287.1NM_032104; NM_032104.2 NP_115286; NP_115286.1 NM_032103; NM_032103.2NP_002472; NP_002472.2 NM_002481; NM_002481.3 MAPK14 NP_620582;NP_620582.1 NM_139013; NM_139013.2 NP_001306; NP_001306.1 NM_001315;NM_001315.2 NP_620583; NP_620583.1 NM_139014; NM_139014.2 NP_620581;NP_620581.1 NM_139012; NM_139012.2 CXCR4 NP_001008540; NP_001008540.1NM_001008540; NM_001008540.1 NP_003458; NP_003458.1 NM_003467;NM_003467.2 MEF2A NP_001165365; NP_001165365.1 NM_001171894;NM_001171894.1 NP_001124400; NP_001124400.1 NM_001130928; NM_001130928.1NP_001124399; NP_001124399.1 NM_001130927; NM_001130927.1 NP_001124398;NP_001124398.1 NM_001130926; NM_001130926.1 NP_005578; NP_005578.2NM_005587; NM_005587.2 TGFB1 NP_000651; NP_000651.3 NM_000660;NM_000660.4 NR3C1 NP_001191194; NP_001191194.1 NM_001204265;NM_001204265.1 NP_001019265; NP_001019265.1 NM_001024094; NM_001024094.1NP_001018661; NP_001018661.1 NM_001020825; NM_001020825.1 NP_001018087;NP_001018087.1 NM_001018077; NM_001018077.1 NP_001018086; NP_001018086.1NM_001018076; NM_001018076.1 NP_001018085; NP_001018085.1 NM_001018075;NM_001018075.1 NP_001018084; NP_001018084.1 NM_001018074; NM_001018074.1NP_000167; NP_000167.1 NM_000176; NM_000176.2 MAP3K5 NP_005914;NP_005914.1 NM_005923; NM_005923.3 CDC42 NP_426359; NP_426359.1NM_044472; NM_044472.2 NP_001782; NP_001782.1 NM_001791; NM_001791.3NP_001034891; NP_001034891.1 NM_001039802; NM_001039802.1 RAF1NP_002871; NP_002871.1 NM_002880; NM_002880.3 CFB NP_001701; NP_001701.2NM_001710; NM_001710.5 ITGB2 NP_000202; NP_000202.2 NM_000211;NM_000211.3 NP_001120963; NP_001120963.1 NM_001127491; NM_001127491.1ATF2 NP_001871; NP_001871.2 NM_001880; NM_001880.2 CREB1 NP_004370;NP_004370.1 NM_004379; NM_004379.3 NP_604391; NP_604391.1 NM_134442;NM_134442.3 MAP2K6 NP_002749; NP_002749.2 NM_002758; NM_002758.3 MAP3K7NP_663306; NP_663306.1 NM_145333; NM_145333.1 NP_663305; NP_663305.1NM_145332; NM_145332.1 NP_663304; NP_663304.1 NM_145331; NM_145331.1NP_003179; NP_003179.1 NM_003188; NM_003188.2 RPS6KA5 NP_004746;NP_004746.2 NM_004755; NM_004755.2 NP_872198; NP_872198.1 NM_182398;NM_182398.1 TRADD NP_003780; NP_003780.1 NM_003789; NM_003789.3 C5NP_001726; NP_001726.2 NM_001735; NM_001735.2 NCR1 NP_004820;NP_004820.1 NM_004829; NM_004829.5 NP_001138929; NP_001138929.1NM_001145457; NM_001145457.1 NP_001138930; NP_001138930.1 NM_001145458;NM_001145458.1 NP_001229285; NP_001229285.1 NM_001242356; NM_001242356.1NP_001229286; NP_001229286.1 NM_001242357; NM_001242357.1 SOCS1NP_003736; NP_003736.1 NM_003745; NM_003745.1 TAGAP NP_687034;NP_687034.1 NM_152133; NM_152133.1 NP_473455; NP_473455.2 NM_054114;NM_054114.3 NP_620165; NP_620165.1 NM_138810; NM_138810.2 PTGS2NP_000954; NP_000954.1 NM_000963; NM_000963.2 PRDM1 NP_001189;NP_001189.2 NM_001198; NM_001198.3 NP_878911; NP_878911.1 NM_182907;NM_182907.2 PLAUR NP_002650; NP_002650.1 NM_002659; NM_002659.3NP_001005376; NP_001005376.1 NM_00100537; NM_001005376.2 FOS NP_005243;NP_005243.1 NM_005252; NM_005252.3 NFKBIZ NP_113607; NP_113607.1NM_031419; NM_031419.3 NP_001005474; NP_001005474.1 NM_001005474;NM_001005474.2 LILRA5 NP_067073; NP_067073.1 NM_021250; NM_021250.2NP_871714; NP_871714.1 NM_181985; NM_181985.2 NP_870994; NP_870994.1NM_181879; NM_181879.2 NP_871715; NP_871715.1 NM_181986; NM_181986.2RIPK2 NP_003812; NP_003812.1 NM_003821; NM_003821.5 LCP2 NP_005556;NP_005556.1 NM_005565; NM_005565.3 LITAF NP_004853; NP_004853.2NM_004862; NM_004862.3 NP_037531; NP_037531.2 NM_013399; NM_013399.2NP_001129945; NP_001129945.1 NM_001136473; NM_001136473.1 NR_024320;NR_024320.1 TNFRSF8 NP_001234; NP_001234.2 NM_001243; NM_001243.3NP_694421; NP_694421.1 NM_152942; NM_152942.2 MEF2D NP_005911;NP_005911.1 NM_005920; NM_005920.2 CDKN1A NP_000380; NP_000380.1NM_000389; NM_000389.2 NP_510867; NP_510867.1 NM_078467; NM_078467.2NP_001207707; NP_001207707.1 NM_001220778; NM_001220778.1 NP_001207706;NP_001207706.1 NM_001220777; NM_001220777.1 CD83 NP_004224; NP_004224.1NM_004233; NM_004233.3 NP_001035370; NP_001035370.1 NM_001040280;NM_001040280.1 NP_001238830; NP_001238830.1 NM_001251901; NM_001251901.1CASP10 NP_116759; NP_116759.2 NM_032977; NM_032977.3 NP_116756;NP_116756.2 NM_032974; NM_032974.4 NP_001221; NP_001221.2 NM_001230;NM_001230.4 NP_116758; NP_116758.1 NM_032976; NM_032976.3 NP_001193471;NP_001193471.1 NM_001206542; NM_001206542.1 NP_001193453; NP_001193453.1NM_001206524; NM_001206524.1 LTB4R NP_858043; NP_858043.1 NM_181657;NM_181657.3 NP_001137391; NP_001137391.1 NM_001143919; NM_001143919.2

Inflammatory markers uniquely increased in CD14⁺CD16⁻ monocytes frompatients having neurodegenerative disorders relative to CD14⁺CD16⁻monocytes from healthy controls are listed in Table 21.

TABLE 21 List of inflammatory markers increased in CD14⁺CD16⁻ monocytesfrom patients having neurodegenerative disorders relative to CD14⁺CD16⁻monocytes from healthy controls Protein sequence (NCBI Accession No.;mRNA sequence (NCBI Accession No.; Marker Version No.) Version No.) CSF1NP_000748; NP_000748.3 NM_000757; NM_000757.5 NP_757351; NP_757351.1NM_172212; NM_172212.2 NP_757349; NP_757349.1 NM_172210; NM_172210.2IL10 NP_000563; NP_000563.1 NM_000572; NM_000572.2 IL1A NP_000566;NP_000566.3 NM_000575; NM_000575.3 HLA-DRA NP_061984; NP_061984.2NM_019111; NM_019111.4 RAC1 NP_061485; NP_061485.1 NM_018890;NM_018890.3 NP_008839; NP_008839.2 NM_006908; NM_006908.4 GRB2NP_987102; NP_987102.1 NM_203506; NM_203506.2 NP_002077; NP_002077.1NM_002086; NM_002086.4 PLA2G4A NP_077734; NP_077734.1 NM_024420;NM_024420.2 GNAS NP_001070956; NP_001070956.1 NM_001077488;NM_001077488.2 NP_001070957; NP_001070957.1 NM_001077489; NM_001077489.2NP_001070958; NP_001070958.1 NM_001077490; NM_001077490.1 NP_057676;NP_057676.1 NM_016592; NM_016592.2 NP_536351; NP_536351.1 NM_080426;NM_080426.2 NP_536350; NP_536350.2 NM_080425; NM_080425.2 NP_000507;NP_000507.1 NM_000516; NM_000516.4 GNB1 NP_002065; NP_002065.1NM_002074; NM_002074.3 TGFB3 NP_003230; NP_003230.1 NM_003239;NM_003239.2 IL6R NP_000556; NP_000556.1 NM_000565; NM_000565.3NP_852004; NP_852004.1 NM_181359; NM_181359.2 NP_001193795;NP_001193795.1 NM_001206866; NM_001206866.1 CXCL3 NP_002081; NP_002081.2NM_002090; NM_002090.2 IL18 NP_001553; NP_001553.1 NM_001562;NM_001562.3 NP_001230140; NP_001230140.1 NM_001243211; NM_001243211.1IL1RN NP_776214; NP_776214.1 NM_173842; NM_173842.2 NP_000568;NP_000568.1 NM_000577; NM_000577.4 NP_776213; NP_776213.1 NM_173841;NM_173841.2 NP_776215; NP_776215.1 NM_173843; NM_173843.2 KEAP1NP_036421; NP_036421.2 NM_012289; NM_012289.3 NP_987096; NP_987096.1NM_203500; NM_203500.1 LIMK1 NP_002305; NP_002305.1 NM_002314;NM_002314.3 NP_001191355; NP_001191355.1 NM_001204426; NM_001204426.1MYC NP_002458; NP_002458.2 NM_002467; NM_002467.4 NFKB1 NP_003989;NP_003989.2 NM_003998; NM_003998.2 SHC1 NP_892113; NP_892113.4NM_183001; NM_183001.4 NP_001123512; NP_001123512.1 NM_001130040;NM_001130040.1 NP_001123513; NP_001123513.1 NM_001130041; NM_001130041.1NP_001189788; NP_001189788.1 NM_001202859; NM_001202859.1 NP_003020;NP_003020.2 NM_003029; NM_003029.4 TLR2 NP_003255; NP_003255.2NM_003264; NM_003264.3 TLR4 NP_612564; NP_612564.1 NM_138554;NM_138554.3 TNFSF14 NP_003798; NP_003798.2 NM_003807; NM_003807.3NP_742011; NP_742011.2 NM_172014; NM_172014.2 AHR NP_001612; NP_001612.1NM_001621; NM_001621.4 BCL3 NP_005169; NP_005169.1 NM_005178;NM_005178.4 CD44 NP_000601; NP_000601.3 NM_000610; NM_000610.3NP_001001389; NP_001001389.1 NM_001001389; NM_001001389.1 NP_001001390;NP_001001390.1 NM_001001390; NM_001001390.1 NP_001001391; NP_001001391.1NM_001001391; NM_001001391.1 NP_001001392; NP_001001392.1 NM_001001392;NM_001001392.1 NP_001189484; NP_001189484.1 NM_001202555; NM_001202555.1NP_001189485; NP_001189485.1 NM_001202556; NM_001202556.1 NP_001189486;NP_001189486.1 NM_001202557; NM_001202557.1 CD81 NP_004347; NP_004347.1NM_004356; NM_004356.3 CD82 NP_002222; NP_002222.1 NM_002231;NM_002231.3 NP_001020015; NP_001020015.1 NM_001024844; NM_001024844.1FCER1A NP_001992; NP_001992.1 NM_002001; NM_002001.2 FCER1G NP_004097;NP_004097.1 NM_004106; NM_004106.1 IL4R NP_000409; NP_000409.1NM_000418; NM_000418.2 NP_001008699; NP_001008699.1 NM_001008699;NM_001008699.1 IL7R NP_002176; NP_002176.2 NM_002185; NM_002185.2 ITGAMNP_000623; NP_000623.2 NM_000632; NM_000632.3 NP_001139280;NP_001139280.1 NM_001145808; NM_001145808.1 JAK3 NP_000206; NP_000206.2NM_000215; NM_000215.3 KLRB1 NP_002249; NP_002249.1 NM_002258;NM_002258.2 LILRB4 NP_001074907; NP_001074907.1 NM_001081438;NM_001081438.1 NP_006838; NP_006838.3 NM_006847; NM_006847.3 PTAFRNP_000943; NP_000943.1 NM_000952; NM_000952.4 NP_001158193;NP_001158193.1 NM_001164721; NM_001164721.1 NP_001158194; NP_001158194.1NM_001164722; NM_001164722.2 NP_001158195; NP_001158195.1 NM_001164723;NM_001164723.2 RUNX1 NP_001745; NP_001745.2 NM_001754; NM_001754.4NP_001001890; NP_001001890.1 NM_001001890; NM_001001890.2 NP_001116079;NP_001116079.1 NM_001122607; NM_001122607.1 SELL NP_000646; NP_000646.2NM_000655; NM_000655.4 TNFSF8 NP_001235; NP_001235.1; NM_001244;NM_001244.3 NP_001239219; NP_001239219.1 NM_001252290; NM_001252290.1TRAF3 NP_663777; NP_663777.1 NM_145725; NM_145725.1 NP_001186356;NP_001186356.1 NM_001199427; NM_001199427.1 NP_003291; NP_003291.2NM_003300; NM_003300.3 NP_663778; NP_663778.1 NM_145726; NM_145726.2CCL2 NP_002973; NP_002973.1 NM_002982; NM_002982.3 CCL4 NP_002975;NP_002975.1 NM_002984; NM_002984.2 CCR1 NP_001286; NP_001286.1NM_001295; NM_001295.2 TLR1 NP_003254; NP_003254.2 NM_003263;NM_003263.3 AAC34137; AAC34137.1 U88540; U88540.1 AAI09094; AAI09094.1BC109093; BC109093.1 AAI09095; AAI09095.1 BC109094; BC109094.1 AAH89403;AAH89403.1 BC089403; BC089403.1 AAY85642; AAY85642.1 DQ012263;DQ012263.1 AAY85640; AAY85640.1 DQ012261; DQ012261.1 AAY85638;AAY85638.1 DQ012259; DQ012259.1 AAY85636; AAY85636.1 DQ012257;DQ012257.1 AAY85634; AAY85634.1 DQ012255; DQ012255.1 AAY85643;AAY85643.1 DQ012264; DQ012264.1 AAY85641; AAY85641.1 DQ012262;DQ012262.1 AAY85639; AAY85639.1 DQ012260; DQ012260.1 AAY85637;AAY85637.1 DQ012258; DQ012258.1 AAY85635; AAY85635.1 DQ012256;DQ012256.1 AAY85633; AAY85633.1 DQ012254; DQ012254.1 TLR5 NP_003259;NP_003259.2 NM_003268; NM_003268.5 AAC34136; AAC34136.1 U88881; U88881.1BAG55042; BAG55042.1 AB445645; AB445645.1 AAI09120; AAI09120.1 BC109119;BC109119.1 AAI09119; AAI09119.1 BC109118; BC109118.1 BAB43955;BAB43955.1 AB060695; AB060695.1

Diagnostic Methods

Provided herein are methods of diagnosing a neurodegenerative disorder.These methods include determining a level of one or more (e.g., at leasttwo, three, four, five or six) microRNAs listed in Tables 1-19 and/orone or more (e.g., at least two, three, four, five or six) inflammatorymarkers listed in Tables 20-21 in cerebrospinal fluid or a CD14⁺CD16⁻ orCD14⁺CD16⁻ monocyte (e.g., peripheral or blood-derived monocyte) fromthe subject, and comparing the level of the one or more microRNAs and/orthe one or more inflammatory markers with a reference level of the oneor more microRNAs and/or one or more inflammatory markers. An increaseor decrease in the level of the one or more microRNAs and/or the levelof the one or more inflammatory markers as compared to the referencelevel(s) indicates that the subject has a neurodegenerative disease asoutlined in detail below.

In some embodiments, a subject can be diagnosed as having aneurodegenerative disorder if the level of one or more or more (e.g., atleast two, three, four, five or six) microRNAs listed in Table 1 in theCSF or a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte (e.g., peripheral orblood-derived monocyte) from the subject is increased compared to areference level of the one or more microRNAs listed in Table 1, and/orif the level of one or more (e.g., at least two, three, four, five orsix) microRNAs listed in Table 2 in the CSF or a CD14⁺CD16⁻ orCD14⁺CD16⁻ monocyte (e.g., peripheral or blood-derived monocyte) fromthe subject is decreased compared to a reference level of the one ormore microRNAs listed in Table 2.

In some embodiments, a subject can be diagnosed as having aneurodegenerative disorder if the level of one or more (e.g., at leasttwo, three, four, five or six) microRNAs listed in Tables 3 and 12 in aCD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte from the subject is increased comparedto a reference level of the one or more (e.g., at least two, three,four, five or six) microRNAs listed in Tables 3 and 12, and/or if thelevel of one or more microRNAs listed in Tables 4 and 13 in a CD14⁺CD16⁻or CD14⁺CD16⁺ monocyte from the subject is decreased compared to areference level of the one or more microRNAs listed in Tables 4 and 13.

In some embodiments, a subject can be diagnosed as having aneurodegenerative disorder if the level of one or more (e.g., at leasttwo, three, four, five or six) microRNAs listed in Table 5 and Table 14,and/or one or more inflammatory markers (e.g., at least two, three,four, five or six) in Table 21 in a CD14⁺CD16⁻ monocyte from the subjectis increased compared to a reference level of the one or more microRNAslisted in Table 5 and Table 14 and/or a reference level of the one ormore inflammatory markers listed in Table 21; and/or if the level of oneor more (e.g., at least two, three, four, five or six) microRNAs listedin Table 6 and Table 15, and/or one or more (e.g., at least two, three,four, five or six) inflammatory markers in Table 20 in a CD14⁺CD16⁻monocyte from the subject is decreased compared to a reference level ofthe one or more microRNAs listed in Table 6 and Table 15 and/or areference level of the one or more inflammatory markers listed in Table20.

In some embodiments, a subject can be diagnosed as having amyotrophiclateral sclerosis if the level of one or more (e.g., at least two,three, four, five or six) microRNAs listed in Tables 5 and 7, and/or oneor more (e.g., at least two, three, four, five or six) inflammatorymarkers listed in Table 21 in a CD14⁺CD16⁻ monocyte from the subject isincreased compared to a reference level of the one or more microRNAslisted in Tables 5 and 7, and/or a reference level of the one or moreinflammatory markers in Table 21; and/or if the level of one or more(e.g., at least two, three, four, five or six) microRNAs listed inTables 6 and 8, and/or one or more (e.g., at least two, three, four,five or six) inflammatory markers listed in Table 20 in a CD14⁺CD16⁻monocyte from the subject is decreased compared to a reference level ofthe one or more microRNAs listed in Tables 6 and 8, and/or a referencelevel of one or more inflammatory markers listed in Table 20.

In some embodiments, a subject can be diagnosed as having amyotrophiclateral sclerosis if the level of one or more (e.g., at least two,three, four, five or six) microRNAs listed in Table 9 in a CD14⁺CD16⁺monocyte from the subject is increased compared to a reference level ofthe one or more microRNAs listed in Table 9, and/or if the level of oneor more (e.g., one, two, or three) microRNAs listed in Table 10 in aCD14⁺CD16⁺ monocyte from the subject is decreased as compared to areference level of the one or more microRNAs listed in Table 10.

In some embodiments, a subject can be diagnosed as having amyotrophiclateral sclerosis if the level of one or more (e.g., at least two,three, four, five or six) of hsa-miR-27b, hsa-miR-99b, hsa-miR-146a,hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p are increased incerebrospinal fluid of the subject compared to a reference level ofhsa-miR-27b, hsa-miR-99b, hsa-miR-146a, hsa-miR-150, hsa-miR-328, andhsa-miR-532-3p.

In some embodiments, a subject can be diagnosed as having familial ALSif the level of hsa-miR-27b in the cerebrospinal fluid from the subjectis increased compared to a reference level of hsa-miR-27b and the levelof one or more (e.g., one, two, three, four, or five) of hsa-miR-99b,hsa-miR-146a, hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p in thecerebrospinal fluid from the subject is decreased or not significantlychanged compared to a reference level of one or more of hsa-miR-99b,hsa-miR-146a, hsa-miR-150, hsa-miR-328, and hsa-miR-532-3p.

In embodiments, a subject can be diagnosed as having sporadic ALS if thelevel of two or more (e.g., two, three, four, five, or six) microRNAsselected from hsa-27b, hsa-miR-99b, hsa-miR-146a, hsa-miR-150,hsa-miR-328, and hsa-miR-532-3p in the cerebrospinal fluid from thesubject is increased compared to a reference level of the one or moremicroRNAs. In embodiments, a subject can be diagnosed as having sporadicALS if the level of one or more (e.g., two, three, four or five)microRNAs selected from hsa-miR-99b, hsa-miR-146a, hsa-miR-150,hsa-miR-328, and hsa-miR-532-3p in the cerebrospinal fluid from thesubject is increased compared to a reference level of the one or moremicroRNAs.

In some embodiments, a subject can be diagnosed as having multiplesclerosis if the level of one or more (e.g., at least two, three, four,five, or six) microRNAs in Table 14 and Table 16, and/or one or more(e.g., at least two, three, four, five, or six) inflammatory markers inTable 21 in a CD14⁺CD16⁻ monocyte from the subject is increased comparedto a reference level of the one or more microRNAs listed in Table 14 andTable 16 and/or the reference level of the one or more inflammatorymarkers listed in Table 21; and/or if the level of one or more (e.g., atleast two, three, four, five, or six) microRNAs in Table 15 and Table17, and/or one or more (e.g., at least two, three, four, five, or six)inflammatory markers in Table 20 in a CD14⁺CD16⁻ monocyte from thesubject is decreased compared to a reference level of the one or moremicroRNAs listed in Table 15 and Table 17, and/or the reference level ofthe one or more inflammatory markers listed in Table 20.

In some embodiments, a subject can be diagnosed as having multiplesclerosis if the level of one or more (e.g., at least two, three, four,five, or six) microRNAs in Table 18 in CD14⁺CD16⁺ monocyte from thesubject is increased compared to a reference level of the one or moremicroRNAs in Table 18 and/or if the level of one or more (e.g., at leasttwo, three, four, five, or six) microRNAs in Table 19 in a CD14⁺CD16⁺monocyte from the subject is decreased compared to a reference level ofthe one or more microRNAs listed in Table 19.

The levels of the one or more microRNAs (both the mature and precursormicroRNAs) described in Tables 1-19 can be determined using molecularbiology methods known in the art. For example, levels of any of themicroRNAs described herein can be measured using techniques that includethe use of a polymerase chain reaction (PCR) and suitable primers, e.g.,quantitative real-time PCR (qRT-PCR). Primers for each of the mature andprecursor microRNAs described herein can be designed using methods knownin the art. Likewise, the levels of an mRNA encoding any of theinflammatory markers in Tables 20 and 21 can be determined usingtechniques that include the use of a PCR and suitable primers. Forexample, a primer can contain at least 7 (e.g., at least 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20) contiguous nucleotides that arecomplementary to a sequence present in the target microRNA or the targetinflammatory marker mRNA. Primers can include one or more of themodifications described herein (e.g., one or more modifications in thebackbone, one or more modifications in the nucleobase(s), and one ormore modifications in the sugar(s)). The primers can also include alabel (e.g., a radioisotope or a fluorophore). The primers can also beconjugated to secondary molecules or agents in order to improve thestability of the primers (as described herein).

The levels of a protein encoded by the inflammatory marker genes listedin Tables 20 and 21 can be detected using a number of techniques knownin the art which utilize antibodies that specifically bind to one of theproteins listed in Tables 20 and 21 (e.g., immunoblotting).

Any of the methods described herein may further include obtaining orcollecting a sample from a subject (e.g., a biological sample containingcerebrospinal fluid or peripheral blood). In some embodiments, themethods (e.g., any of the methods described herein) further includepurifying a monocyte (e.g., a CD14⁺CD16⁻ monocyte or a CD14⁺CD16⁺monocyte from a biological sample from the subject). Methods ofpurifying a CD14⁺CD16⁻ monocyte or a CD14⁺CD16⁺ monocyte can beperformed using a variety of methods known in the art, e.g.,antibody-based methods, such as fluorescence-assisted cell sorting(FACS).

Any of the methods described herein can be performed on patientspresenting to a health care facility (e.g., a hospital, clinic, or anassisted care facility). The subjects may present with one or moresymptoms of a neurodegenerative disorder (e.g., any of the symptoms of aneurodegenerative disorder described herein). The subject can alsopresent with no symptoms (an asymptomatic subject) or just one symptomof a neurodegenerative disorder. The subject can have a familial historyof a neurodegenerative disorder (e.g., familial ALS).

The diagnostic methods described herein can be performed by any healthcare professional (e.g., a physician, a laboratory technician, a nurse,a physician's assistant, and a nurse's assistant). The diagnosticmethods described herein can be used in combination with any additionaldiagnostic testing methods known in the art (e.g., the observation orassessment of one or more symptoms of a neurodegenerative disorder in asubject).

Methods of Selecting a Subject for Treatment

Also provided are methods of selecting a subject for treatment of aneurodegenerative disorder. These methods include determining a level ofone or more (e.g., at least two, three, four, five, or six) of themicroRNAs listed in Tables 1-19 and/or the level of one or more (e.g.,at least two, three, four, five, or six) inflammatory markers listed inTables 20 and 21 in cerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺monocyte from the subject; comparing the level of the one or moremicroRNAs in cerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁻ monocytefrom the subject to a reference level of the one or more microRNAsand/or a reference level of the one or more inflammatory markers; andselecting a subject having an increase in the level of one or more(e.g., at least two, three, four, five, or six) microRNAs listed inTables 1, 3, 5, 7, 9, 11, 12, 14, 16, or 18, and/or one or more (e.g.,at least two, three, four, five, or six) inflammatory markers listed inTable 21 in cerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocytefrom the subject compared to a reference level of the one or moremicroRNAs listed in Tables 1, 3, 5, 7, 9, 11, 12, 14, 16, or 18, and/ora reference level of the one or more inflammatory markers listed inTable 21; and/or a decrease in the level of one or more (e.g., at leasttwo, three, four, five, or six) microRNAs listed in Tables 2, 4, 6, 8,10, 13, 15, 17, and 19, and/or a one or more (e.g., at least two, three,four, five, or six) inflammatory markers listed in Table 20 incerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte from thesubject compared to a reference level of the one or more microRNAslisted in Tables 2, 4, 6, 8, 10, 13, 15, 17, and 19, and/or a referencelevel of one or more inflammatory markers listed in Table 20 fortreatment of a neurodegenerative disorder.

A subject may be selected for treatment on the basis of the relativeexpression of one or more (e.g., at least two, three, four, five, orsix) microRNAs listed in Tables 1-19 and/or one or more (e.g., at leasttwo, three, four, five, or six) inflammatory markers listed in Tables 20and 21 as described in the above section describing diagnostic methods.For example, an increase in the level of one or more (e.g., at leasttwo, three, four, five, or six) microRNAs listed in Tables 1, 3, 5, 7,9, 11, 12, 14, 16, or 18 and/or the level of one or more (e.g., at leasttwo, three, four, five, or six) inflammatory markers listed in Table 21(compared to a reference level), as used to diagnose a subject as havinga neurodegenerative disorder, may likewise be used to select a subjectfor treatment of a neurodegenerative disorder. Similarly, a decrease inthe level of one or more (e.g., at least two, three, four, five, or six)microRNAs listed in Tables 2, 4, 6, 8, 10, 13, 15, 17, and 19 and/or thelevel of one or more (e.g., at least two, three, four, five, or six)inflammatory markers listed in Table 20 (compared to a reference level),as used to diagnose a subject as having a neurodegenerative disorder,may likewise be used to select a subject for treatment of aneurodegenerative disorder.

The levels of the one or more microRNAs (both the mature and precursormicroRNAs) described in Tables 1-19 can be determined using molecularbiology methods known in the art. For example, levels of any of themicroRNAs described herein can be measured using techniques that includethe use of a polymerase chain reaction (PCR) and suitable primers, e.g.,quantitative real-time PCR (qRT-PCR). Primers for each of the mature andprecursor microRNAs described herein can be designed using methods knownin the art. Likewise, the levels of an mRNA encoding any of theinflammatory markers in Tables 20 and 21 can be determined usingtechniques that include the use of a PCR and suitable primers. Forexample, a primer can contain at least 7 (e.g., at least 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20) contiguous nucleotides that arecomplementary to a sequence present in the target microRNA or the targetinflammatory marker mRNA. Primers can include one or more of themodifications described herein (e.g., one or more modifications in thebackbone, one or more modifications in the nucleobase(s), and one ormore modifications in the sugar(s)). The primers can also include alabel (e.g., a radioisotope or a fluorophore). The primers can also beconjugated to secondary molecules or agents in order to improve thestability of the primers (as described herein). The methods can beperformed by any health care professional (e.g., a physician, a nurse, aphysician's assistant, a laboratory technician, or a nurse's assistant).

The subjects may present with one or more symptoms (e.g., at least two,three, or four) of a neurodegenerative disorder (e.g., any of thesymptoms of a neurodegenerative disorder described herein). The subjectcan also present with no symptoms or just one symptom of aneurodegenerative disorder. The subject can have a familial history of aneurodegenerative disorder (e.g., familial ALS). The subject can bepreviously diagnosed as having a neurodegenerative disorder.

Treatments of neurodegenerative disorders that can be administered tothe subject include riluzole, corticosteroids, beta-interferon,glatiramer, fingolimod, natalizumab, mitoxantrone, muscle relaxants, andamantadine. Additional treatments of neurodegenerative disorders includephysical therapy and plasmapheresis.

Methods of Identifying a Subject at Risk of Developing aNeurodegenerative Disorder

Also provided are methods of identifying a subject at risk of developinga neurodegenerative disorder. These methods include determining a levelof one or more (e.g., at least two, three, four, five, or six) microRNAslisted in Tables 1-19 and/or one or more (e.g., at least two, three,four, five, or six) inflammatory markers listed in Tables 20 and 21 inthe cerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte from thesubject; comparing the level of the one or more microRNAs in thecerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte from thesubject to a reference level of the one or more microRNAs and/or areference level of the one or more inflammatory markers. A subject isidentified as having an increased risk of developing a neurologicaldisorder if the level of one or more (e.g., at least two, three, four,five, or six) microRNAs listed in Tables 1, 3, 5, 7, 9, 11, 12, 14, 16,or 18, and/or one or more (e.g., at least two, three, four, five, orsix) inflammatory markers listed in Table 21 in cerebrospinal fluid or aCD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte from the subject is increased comparedto a reference level of the one or more microRNAs listed in Tables 1, 3,5, 7, 9, 11, 12, 14, 16, or 18, and/or a reference level of the one ormore inflammatory markers listed in Table 21; and/or the level of one ormore (e.g., at least two, three, four, five, or six) microRNAs listed inTables 2, 4, 6, 8, 10, 13, 15, 17, and 19, and/or a one or more (e.g.,at least two, three, four, five, or six) inflammatory markers listed inTable 20 in cerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocytefrom the subject is decreased compared to a reference level of the oneor more microRNAs listed in Tables 2, 4, 6, 8, 10, 13, 15, 17, and 19,and/or a reference level of one or more inflammatory markers listed inTable 20.

In some embodiments, a subject is identified having a decreased risk ofdeveloping a neurological disorder if the level of one or more (e.g., atleast two, three, four, five, or six) microRNAs listed in Tables 1, 3,5, 7, 9, 11, 12, 14, 16, or 18, and/or one or more (e.g., at least two,three, four, five, or six) inflammatory markers listed in Table 21 incerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte from thesubject is decreased or not significantly changed compared to areference level of the one or more microRNAs listed in Tables 1, 3, 5,7, 9, 11, 12, 14, 16, or 18, and/or a reference level of the one or moreinflammatory markers listed in Table 21; and/or the level of one or more(e.g., at least two, three, four, five, or six) microRNAs listed inTables 2, 4, 6, 8, 10, 13, 15, 17, and 19, and/or a one or more (e.g.,at least two, three, four, five, or six) inflammatory markers listed inTable 20 in cerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocytefrom the subject is increased or not significantly changed compared to areference level of the one or more microRNAs listed in Tables 2, 4, 6,8, 10, 13, 15, 17, and 19, and/or a reference level of one or moreinflammatory markers listed in Table 20.

In some embodiments, a subject may be identified as having an increasedor decreased risk of developing a neurodegenerative disorder on thebasis of the relative expression of one or more (e.g., at least two,three, four, five, or six) microRNAs listed in Tables 1-19 and/or one ormore (e.g., at least two, three, four, five, or six) inflammatorymarkers listed in Tables 20 and 21 in the cerebrospinal fluid or aCD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte from the subject as compared to areference value as described in the above section describing diagnosticmethods. For example, an increase in the level of one or more (e.g., atleast two, three, four, five, or six) microRNAs listed in Tables 1, 3,5, 7, 9. 11, 12, 14, 16, and 18 and/or the level of one or more (e.g.,at least two, three, four, five, or six) inflammatory markers listed inTable 21 in the cerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺monocyte from the subject (compared to a reference level), as used todiagnose a subject as having a neurodegenerative disorder, may likewisebe used to identify a subject at increased risk of developing aneurodegenerative disorder. Similarly, a decrease in the level of one ormore (e.g., at least two, three, four, five, or six) microRNAs listed inTables 2, 4, 6, 8, 10, 13, 15, 17, and 19 or the level of one or more(e.g., at least two, three, four, five, or six) inflammatory markerslisted in Table 20 in the cerebrospinal fluid or a CD14⁺CD16⁻ orCD14⁺CD16⁺ monocyte (compared to a reference level), as used to diagnosea subject as having a neurodegenerative disorder, may likewise be usedto identify a subject at increased risk of developing aneurodegenerative disorder.

In some embodiments, an increase in the level of one or more (e.g., atleast two, three, four, five, or six) microRNAs listed in Tables 2, 4,6, 8, 10, 13, 15, 17, or 19 and/or the level of one or more (e.g., atleast two, three, four, five, or six) inflammatory markers listed inTable 20 in the cerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺monocyte from the subject (compared to a reference level), when adecrease in the level of the one or more microRNAs or a decrease in thelevel of the one or more inflammatory markers indicates a diagnosis of aneurodegenerative disease (as detailed in the section describingdiagnostic methods above), indicates that the subject is at decreasedrisk of developing a neurodegenerative disorder. In some embodiments, adecrease in the level of one or more (e.g., at least two, three, four,five, or six) microRNAs listed in Tables 1, 3, 5, 7, 9, 11, 12, 14, 16,and 18, or the level of one or more (e.g., at least two, three, four,five, or six) inflammatory markers listed in Table 21 in thecerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte from thesubject (compared to a reference level), when an increase in the levelof one or more microRNAs or an increase in the level of the one or moreinflammatory markers indicates a diagnosis of a neurodegenerativedisorder (as detailed in the section describing diagnostic methodsabove), indicates that the subject is at decreased risk of developing aneurodegenerative disorder.

In any of the methods described herein, the increased or decreased riskis relative to a subject that does not have an increase or decrease inthe levels of one or more (e.g., at least two, three, four, five, orsix) microRNA listed in Tables 1-19 and/or does not have an increase ordecrease in the levels of one or more (e.g., at least two, three, four,five, or six) inflammatory markers listed in Tables 20 and 21 (e.g., asubject that is not diagnosed as having a neurodegenerative disorderusing any of the methods described herein).

The levels of any of the microRNAs in Tables 1-19 or the levels of anyof the inflammatory markers listed in Tables 20 and 21 may be performedusing standard molecular biology methods (e.g., the PCR-based andantibody-based methods described herein). The methods can be performedby any health care professional (e.g., a physician, a nurse, aphysician's assistant, a laboratory technician, or a nurse's assistant).

The subjects may present with one or more symptoms of aneurodegenerative disorder (e.g., any of the symptoms of aneurodegenerative disorder described herein). The subject can alsopresent with no symptoms or just one symptom of a neurodegenerativedisorder. The subject can have a family history of a neurodegenerativedisorder (e.g., familial ALS). Subjects identified as having anincreased risk of developing a neurodegenerative disease may beadministered a treatment for a neurodegenerative disorder or may beadministered a new or alternative treatment for a neurodegenerativedisorder. Subjects identified as having an increased risk of developinga neurodegenerative disorder can also undergo more aggressivetherapeutic treatment (e.g., increased periodicity of clinic or hospitalvisits).

Methods of Predicting the Rate of Disease Progression

Also provided are methods of predicting the rate of disease progressionin a subject having a neurodegenerative disorder. These methods includedetermining a level of one or more (e.g., at least two, three, four,five, or six) microRNAs listed in Tables 1-19 and/or one or more (e.g.,at least two, three, four, five, or six) inflammatory markers listed inTables 20 and 21 in the cerebrospinal fluid or a CD14⁺CD16⁻ orCD14⁺CD16⁺ monocyte from the subject; comparing the level of the one ormore microRNAs and/or the one or more inflammatory markers to areference level of the one or more microRNAs and/or a reference level ofthe one or more inflammatory markers. A subject is predicted to have anincreased rate of disease progression if the level of one or more (e.g.,at least two, three, four, five, or six) microRNAs listed in Tables 1,3, 5, 7, 9, 11, 12, 14, 16, or 18, and/or one or more (e.g., at leasttwo, three, four, five, or six) inflammatory markers listed in Table 21in cerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte from thesubject is increased compared to a reference level of the one or moremicroRNAs listed in Tables 1, 3, 5, 7, 9, 11, 12, 14, 16, or 18, and/ora reference level of the one or more inflammatory markers listed inTable 21; and/or the level of one or more (e.g., at least two, three,four, five, or six) microRNAs listed in Tables 2, 4, 6, 8, 10, 13, 15,17, and 19, and/or a one or more (e.g., at least two, three, four, five,or six) inflammatory markers listed in Table 20 in cerebrospinal fluidor a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte from the subject is decreasedcompared to a reference level of the one or more microRNAs listed inTables 2, 4, 6, 8, 10, 13, 15, 17, and 19, and/or a reference level ofone or more inflammatory markers listed in Table 20.

In some embodiments, a subject is predicted to have a slower or averagerate of disease progression if the level of one or more (e.g., at leasttwo, three, four, five, or six) microRNAs listed in Tables 1, 3, 5, 7,9, 11, 12, 14, 16, or 18, and/or one or more (e.g., at least two, three,four, five, or six) inflammatory markers listed in Table 21 incerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte from thesubject is decreased or not significantly changed compared to areference level of the one or more microRNAs listed in Tables 1, 3, 5,7, 9, 11, 12, 14, 16, or 18, and/or a reference level of the one or moreinflammatory markers listed in Table 21; and/or the level of one or more(e.g., at least two, three, four, five, or six) microRNAs listed inTables 2, 4, 6, 8, 10, 13, 15, 17, and 19, and/or a one or more (e.g.,at least two, three, four, five, or six) inflammatory markers listed inTable 20 in cerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocytefrom the subject is increased or not significantly changed compared to areference level of the one or more microRNAs listed in Tables 2, 4, 6,8, 10, 13, 15, 17, and 19, and/or a reference level of one or moreinflammatory markers listed in Table 20.

In some embodiments, a subject may be predicted to have an increased ordecreased rate of disease progression on the basis of the relativeexpression of one or more (e.g., at least two, three, four, five, orsix) microRNAs listed in Tables 1-19 and/or one or more (e.g., at leasttwo, three, four, five, or six) inflammatory markers listed in Tables 20and 21 in the cerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocytefrom the subject as compared to a reference value as described in theabove section describing diagnostic methods. For example, an increase inthe level of one or more (e.g., at least two, three, four, five, or six)microRNAs listed in Tables 1, 3, 5, 7, 9, 11, 12, 14, 16, and 18, and/orthe level of one or more (e.g., at least two, three, four, five, or six)inflammatory markers listed in Table 21 in the cerebrospinal fluid or aCD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte from the subject (compared to areference level), as used to diagnose a subject as having aneurodegenerative disorder, may likewise be used to predict an increasedrate of disease progression. Similarly, a decrease in the level of oneor more (e.g., at least two, three, four, five, or six) microRNAs listedin Tables 2, 4, 6, 8, 10, 13, 15, 17, and 19 and/or the level of one ormore (e.g., at least two, three, four, five, or six) inflammatorymarkers listed in Table 20 in the cerebrospinal fluid or a CD14⁺CD16⁻ orCD14⁺CD16⁺ monocyte (compared to a reference level), as used to diagnosea subject as having a neurodegenerative disorder, may likewise be usedto predict an increased rate of disease progression.

In some embodiments, an increase in the level of one or more (e.g., atleast two, three, four, five, or six) microRNAs listed in Tables 2, 4,6, 8, 10, 13, 15, 17, and 19, and/or the level of one or more (e.g., atleast two, three, four, five, or six) inflammatory markers in Table 20in the cerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte fromthe subject (compared to a reference level), when a decrease in thelevel of the one or more microRNAs and/or a decrease in the level of theone or more inflammatory markers indicates a diagnosis of aneurodegenerative disease (as detailed in the section describingdiagnostic methods above), can be used to predict a decreased or averagerate of disease progression. In some embodiments, a decrease in thelevel of one or more (e.g., at least two, three, four, five, or six)microRNAs listed in Tables 1, 3, 5, 7, 9, 11, 12, 14, 16, or 18, and/orthe level of one or more (e.g., at least two, three, four, five, or six)inflammatory markers listed in Table 21 in the cerebrospinal fluid or aCD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte from the subject (compared to areference level), when an increase in the level of the one or moremicroRNAs and/or an increase in the level of the one or moreinflammatory markers indicates a diagnosis of a neurodegenerativedisorder (as detailed in the section describing diagnostic methodsabove), can be used to predict a decreased or average rate of diseaseprogression.

In some embodiments, the rate of disease progression is the rate ofonset of one or more (e.g., one, two, three, or four) symptoms (e.g.,ataxia) of a neurodegenerative disorder, the rate of increasingintensity (worsening) of symptoms of a neurodegenerative disorder, thefrequency of one or more symptoms of a neurodegenerative disorder, theduration of one or more symptoms of a neurodegenerative disorder, or thelongevity of the subject. For example, an increase in the rate ofdisease progression can be manifested by one or more of: an increase inthe rate of onset of one or more (new) symptoms of a neurodegenerativedisorder, an increase in the rate of increasing intensity (worsening) ofone or more symptoms of a neurodegenerative disorder, an increase in theduration of one or more symptoms of a neurodegenerative disorder, and adecrease in the longevity of the subject.

The rate of disease progression determined using the methods describedherein can be compared to the rate of disease progression in subjectsthat do not have an increase or a decrease in the level of the one ormore (e.g., at least two, three, four, five, or six) microRNAs listed inTables 1-19 and/or do not have an increase or a decrease in the level ofthe one or more (e.g., at least two, three, four, five, or six)inflammatory markers listed in Tables 20 and 21 in their CSF or in aCD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte. In some embodiments, the rate ofdisease progression can be compared to the average rate of diseaseprogression for all subjects diagnosed as having the sameneurodegenerative disease.

The levels of any of the microRNAs in Tables 1-19 or the levels of anyof the inflammatory markers listed in Tables 20 and 21 may be performedusing standard molecular biology methods (e.g., the PCR-based andantibody-based methods described herein). The methods can be performedby any health care professional (e.g., a physician, a nurse, aphysician's assistant, a laboratory technician, or a nurse's assistant).

The subjects may present with one or more (e.g., one, two, three, orfour) symptoms of a neurodegenerative disorder (e.g., any of thesymptoms of a neurodegenerative disorder described herein). The subjectcan also present with no symptoms or just one symptom of aneurodegenerative disorder. The subject can have a family history of aneurodegenerative disorder (e.g., familial ALS). In some embodiments,the subject can already be diagnosed as having a neurodegenerativedisorder.

Some embodiments of these methods further include administering atreatment to a subject predicted to have an increased rate of diseaseprogression. In some embodiments, a subject predicted to have anincreased rate of disease progression is administered a more aggressivetreatment (e.g., increased periodicity of clinic visits).

Methods of Selecting a Subject for Participation in a Clinical Study

Also provided are methods for selecting a subject for participation in aclinical study. These methods include determining a level of one or more(e.g., at least two, three, four, five, or six) microRNAs listed inTables 1-19 and/or one or more (e.g., at least two, three, four, five,or six) inflammatory markers listed in Tables 20 and 21 in thecerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte from thesubject; comparing the level of the one or more microRNAs and/or thelevel of the one or more inflammatory markers to a reference level ofthe one or more microRNAs and/or a reference level of the one or moreinflammatory markers, and selecting a subject having an increase ordecrease in the level of the one or more microRNAs and/or one or moreinflammatory markers in the cerebrospinal fluid or a CD14⁺CD16⁻ orCD14⁺CD16⁻ monocyte from the subject compared to the reference level (asdescribed in detail below) for participation in a clinical study. Insome embodiments, a subject is selected for participation in a clinicalstudy if the level of one or more (e.g., at least two, three, four,five, or six) microRNAs listed in Tables 1, 3, 5, 7, 9, 11, 12, 14, 16,or 18, and/or one or more (e.g., at least two, three, four, five, orsix) inflammatory markers listed in Table 21 in cerebrospinal fluid or aCD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte from the subject is increased comparedto a reference level of the one or more microRNAs listed in Tables 1, 3,5, 7, 9, 11, 12, 14, 16, or 18, and/or a reference level of the one ormore inflammatory markers listed in Table 21; and/or the level of one ormore (e.g., at least two, three, four, five, or six) microRNAs listed inTables 2, 4, 6, 8, 10, 13, 15, 17, and 19, and/or a one or more (e.g.,at least two, three, four, five, or six) inflammatory markers listed inTable 20 in cerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocytefrom the subject is decreased compared to a reference level of the oneor more microRNAs listed in Tables 2, 4, 6, 8, 10, 13, 15, 17, and 19,and/or a reference level of one or more inflammatory markers listed inTable 20.

In some embodiments, a subject is selected for participation in aclinical study if the level of one or more (e.g., at least two, three,four, five, or six) microRNAs listed in Tables 1, 3, 5, 7, 9, 11, 12,14, 16, or 18, and/or one or more (e.g., at least two, three, four,five, or six) inflammatory markers listed in Table 21 in cerebrospinalfluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte from the subject isdecreased or not significantly changed compared to a reference level ofthe one or more microRNAs listed in Tables 1, 3, 5, 7, 9, 11, 12, 14,16, or 18, and/or a reference level of the one or more inflammatorymarkers listed in Table 21; and/or the level of one or more (e.g., atleast two, three, four, five, or six) microRNAs listed in Tables 2, 4,6, 8, 10, 13, 15, 17, and 19, and/or a one or more (e.g., at least two,three, four, five, or six) inflammatory markers listed in Table 20 incerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte from thesubject is increased or not significantly changed compared to areference level of the one or more microRNAs listed in Tables 2, 4, 6,8, 10, 13, 15, 17, and 19, and/or a reference level of one or moreinflammatory markers listed in Table 20.

In some embodiments, a subject can be selected for participation in aclinical study on the basis of the relative expression of one or more(e.g., at least two, three, four, five, or six) microRNAs listed inTables 1-19 and/or one or more (e.g., at least two, three, four, five,or six) inflammatory markers listed in Tables 20 and 21 in thecerebrospinal fluid or a CD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte from thesubject as compared to a reference value as described in the abovesection describing diagnostic methods. For example, an increase in thelevel of one or more (e.g., at least two, three, four, five, or six)microRNAs listed in Tables 1, 3, 5, 7, 9, 11, 12, 14, 16, and 18, and/orthe level of one or more (e.g., at least two, three, four, five, or six)inflammatory markers listed in Table 21 in the cerebrospinal fluid or aCD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte from the subject (compared to areference level), as used to diagnose a subject as having aneurodegenerative disorder, may likewise be used to select a subject forparticipation in a clinical study. Similarly, a decrease in the level ofone or more (e.g., at least two, three, four, five, or six) microRNAslisted in Tables 2, 4, 6, 8, 10, 13, 15, 17, or 19 and/or the level ofone or more (e.g., at least two, three, four, five, or six) inflammatorymarkers listed in Table 20 in the cerebrospinal fluid or a CD14⁺CD16⁻ orCD14⁺CD16⁺ monocyte (compared to a reference level), as used to diagnosea subject as having a neurodegenerative disorder, may likewise be usedto select a subject for participation in a clinical study.

In some embodiments, an increase or no significant change in the levelof one or more (e.g., at least two, three, four, five, or six) microRNAslisted in Tables 2, 4, 6, 8, 10, 13, 15, 17, and 19 and/or the level ofone or more (e.g., at least two, three, four, five, or six) inflammatorymarkers listed in Table 20 in the cerebrospinal fluid or a CD14⁺CD16⁻ orCD14⁺CD16⁻ monocyte from the subject (compared to a reference level),when a decrease in the level of the one or more microRNAs and/or adecrease in the level of the one or more inflammatory markers indicatesa diagnosis of a neurodegenerative disease (as detailed in the sectiondescribing diagnostic methods above), can be used to select a subjectfor participation in a clinical study (e.g., as a control subject). Insome embodiments, a decrease or no significant change in the level ofone or more (e.g., at least two, three, four, five, or six) microRNAslisted in Tables 1, 3, 5, 7, 9, 11, 12, 14, 16, or 18 and/or the levelof one or more (e.g., at least two, three, four, five, or six)inflammatory markers listed in Table 21 in the cerebrospinal fluid or aCD14⁺CD16⁻ or CD14⁺CD16⁺ monocyte from the subject (compared to areference level), when an increase in the level of the one or moremicroRNAs and/or an increase in the level of the one or moreinflammatory markers indicates a diagnosis of a neurodegenerativedisorder (as detailed in the section describing diagnostic methodsabove), can be used to select a subject for participation in a clinicalstudy.

The levels of any of the microRNAs in Tables 1-19 or the levels of anyof the inflammatory markers listed in Tables 20 and 21 may be performedusing standard molecular biology methods (e.g., the PCR-based andantibody-based methods described herein). The methods can be performedby any health care professional (e.g., a physician, a nurse, aphysician's assistant, a laboratory technician, or a nurse's assistant).

In some embodiments, the subject may present with one or more symptomsof a neurodegenerative disorder (e.g., any of the symptoms of aneurodegenerative disorder described herein). In some embodiments, thesubject can also present with no symptoms or just one symptom of aneurodegenerative disorder. In some embodiments, the subject can have afamilial history of a neurodegenerative disorder (e.g., familial ALS).In some embodiments, the subject can already be diagnosed as having aneurodegenerative disorder.

Methods of Treatment

Also provided are methods of treating a neurodegenerative disorder thatinclude administering to a subject at least one (e.g., at least two,three, four, five, or six) agent (e.g., a nucleic acid) that decreasesthe level or activity of one or more (e.g., at least two, three, four,five, or six) of the microRNAs listed in Tables 1, 3, 5, 7, 9, 11, 12,14, 16, or 18 (e.g., an inhibitory nucleic acid, e.g., an antagomir),and/or increases the level or activity of one or more (e.g., at leasttwo, three, four, five, or six) of the microRNAs listed in Tables 2, 4,6, 8, 10, 13, 15, 17, or 19 (e.g., a sense nucleic acid). Also providedare methods of treating a neurodegenerative disorder (e.g., ALS or MS)that include administering to a subject at least one (e.g., at leasttwo, three, four, five, or six) agent (e.g., a nucleic acid) thatdecreases the expression (e.g., protein or mRNA) or activity of one ormore (e.g., at least two, three, four, five, or six) of the inflammatorymarkers listed in Table 21 (e.g., an inhibitory nucleic acid orantibody) and/or increases the expression (e.g., protein or mRNA) and/oractivity of one or more (e.g., at least two, three, four, five, or six)of the genes listed in Table 20 (e.g., a sense nucleic acid). In someembodiments, the subject is first identified or selected for treatmentusing any of the diagnostic methods described herein or any of themethods of predicting a subject at risk of developing aneurodegenerative disorder described herein.

In some embodiments, the subject is administered at least one inhibitorynucleic acid comprising a sequence that is complementary to a contiguoussequence present in hsa-miR-155 (e.g., a contiguous sequence present inmature or precursor hsa-miR-155). In non-limiting embodiments, theinhibitory nucleic acid can be an antisense oligonucleotide, a ribozyme,an siRNA, or an antagomir. In some embodiments, the at least oneinhibitory nucleic acid is injected into the cerebrospinal fluid of asubject. In some embodiments, the injection is intracranial injection orintrathecal injection. In some embodiments, the at least one inhibitorynucleic acid is complexed with one or more cationic polymers and/orcationic lipids (e.g., any of the cationic polymers described herein orknown in the art). Antagomirs to decrease the expression and/or activityof a specific target miRNA (e.g., hsa-miR-155) can be designed usingmethods known in the art (see, e.g., Krutzfeld et al., Nature438:685-689, 2005). Additional exemplary methods for designing andmaking antagomirs and other types of inhibitory nucleic acids aredescribed herein.

In some embodiments, the inhibitory nucleic acid that decreases miR-155levels is the antogmir-155 LNA sequence +TC+AC+A+A+TTA+G+C+AT+T+A (SEQID NO: 262) (wherein the + indicates the presence of an LNA moiety).Methods for designing antagomirs to target microRNA molecules aredescribed in Obad et al., Nature Genetics 43:371-378, 2011. Additionalinhibitory nucleic acids for decreasing the levels or expression ofhsa-miR-155 are described in Worm et al., Nucleic Acids Res.37:5784-5792, 2009, and Murugaiyan et al., J. Immunol. 187:2213-2221,2011.

A subject can be administered at least one (e.g., at least 2, 3, 4, or5) dose of the agent (e.g., one or more inhibitory nucleic acids). Theagent (e.g., one or more inhibitory nucleic acids) can be administeredto the subject at least once a day (e.g., twice a day, three times aday, and four times a day), at least once a week (e.g., twice a week,three times a week, four times a week), and/or at least once a month. Asubject can be treated (e.g., periodically administered the agent) for aprolonged period of time (e.g., at least one month, two months, sixmonths, one year, two years, three years, four years, or five years). Asdescribed in detail herein, the dosage of the agent to be administeredto the subject can be determined by a physician by consideration of anumber of physiological factors including, but not limited to, the sexof the subject, the weight of the subject, the age of the subject, andthe presence of other medical conditions. The agent can be administeredto the subject orally, intravenously, intraarterially, subcutaneously,intramuscularly, intracranially, or via injection into the cerebrospinalfluid. Likewise, the agent may be formulated as a solid (e.g., for oraladministration) or a physiologically acceptable liquid carrier (e.g.,saline) (e.g., for intravenous, intraarterial, subcutaneous,intramuscular, cerebrospinal (intrathecal), or intracranialadministration). In some embodiments, the agent (e.g., one or moreinhibitory nucleic acids) can be administered by injection or can beadministered by infusion over a period of time.

The agents to be administered to a subject for treatment of aneurodegenerative disorder are described below, and can be used in anycombination (e.g., at least one, two, three, four, or five of anycombination of the agents or classes of agents described below).

Inhibitory Nucleic Acids

Inhibitory agents useful in the methods of treatment described hereininclude inhibitory nucleic acid molecules that decrease the expressionor activity of any of the microRNAs (e.g., mature microRNA or precursormicroRNA) listed in Tables 1, 3, 5, 7, 9, 11, 12, 14, 16, or 18 (e.g.,hsa-miR-155), or decrease the expression or activity of any of the mRNAsencoding an inflammatory marker listed in Table 21 (the target mRNA).

Inhibitory nucleic acids useful in the present methods and compositionsinclude antisense oligonucleotides, ribozymes, external guide sequence(EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNAinterference (RNAi) compounds, such as siRNA compounds, modifiedbases/locked nucleic acids (LNAs), antagomirs, peptide nucleic acids(PNAs), and other oligomeric compounds, or oligonucleotide mimeticswhich hybridize to at least a portion of the target nucleic acid andmodulate its function. In some embodiments, the inhibitory nucleic acidsinclude antisense RNA, antisense DNA, chimeric antisenseoligonucleotides, antisense oligonucleotides comprising modifiedlinkages, interference RNA (RNAi), short interfering RNA (siRNA); amicro, interfering RNA (miRNA); a small, temporal RNA (stRNA); or ashort, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa);small activating RNAs (saRNAs), or combinations thereof. See, e.g., WO2010/040112.

In some embodiments, the inhibitory nucleic acids are 10 to 50, 13 to50, or 13 to 30 nucleotides in length. One having ordinary skill in theart will appreciate that this embodies oligonucleotides having antisenseportions of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or any rangetherewithin. In some embodiments, the oligonucleotides are 15nucleotides in length. In some embodiments, the antisense oroligonucleotide compounds of the invention are 12 or 13 to 30nucleotides in length. One having ordinary skill in the art willappreciate that this embodies inhibitory nucleic acids having antisenseportions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 nucleotides in length, or any range therewithin.

In some embodiments, the inhibitory nucleic acids are chimericoligonucleotides that contain two or more chemically distinct regions,each made up of at least one nucleotide. These oligonucleotidestypically contain at least one region of modified nucleotides thatconfers one or more beneficial properties (such as, for example,increased nuclease resistance, increased uptake into cells, increasedbinding affinity for the target) and a region that is a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimericinhibitory nucleic acids of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides, and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures comprise, but are not limited to, U.S. PatentNos. 5,013,830; 5,149,797; 5, 220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,each of which is herein incorporated by reference.

In some embodiments, the inhibitory nucleic acid comprises at least onenucleotide modified at the 2′ position of the sugar, most preferably a2′-O-alkyl, 2′-O-alkyl-O-alkyl or 2′-fluoro-modified nucleotide. Inother preferred embodiments, RNA modifications include 2′-fluoro,2′-amino, and 2′ O-methyl modifications on the ribose of pyrimidines,abasic residues, or an inverted base at the 3′ end of the RNA. Suchmodifications are routinely incorporated into oligonucleotides and theseoligonucleotides have been shown to have a higher Tm (i.e., highertarget binding affinity) than 2′-deoxyoligonucleotides against a giventarget.

A number of nucleotide and nucleoside modifications have been shown tomake the oligonucleotide into which they are incorporated more resistantto nuclease digestion than the native oligodeoxynucleotide—the modifiedoligos survive intact for a longer time than unmodifiedoligonucleotides. Specific examples of modified oligonucleotides includethose comprising modified backbones, for example, phosphorothioates,phosphotriesters, methyl phosphonates, short-chain alkyl or cycloalkylintersugar linkages, or short-chain heteroatomic or heterocyclicintersugar linkages. Most preferred are oligonucleotides withphosphorothioate backbones and those with heteroatom backbones,particularly CH2—NH—O—CH2, CH,˜N(CH3)˜O˜CH2 (known as amethylene(methylimino) or MMI backbone], CH2—O—N (CH3)—CH2, CH2—N(CH3)—N (CH3)—CH2 and O—N (CH3)—CH2—CH2 backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH,); amide backbones(see De Mesmaeker et al., Ace. Chem. Res. 28:366-374, 1995); morpholinobackbone structures (see U.S. Pat. No. 5,034,506); peptide nucleic acid(PNA) backbone (wherein the phosphodiester backbone of theoligonucleotide is replaced with a polyamide backbone, the nucleotidesbeing bound directly or indirectly to the aza nitrogen atoms of thepolyamide backbone, see Nielsen et al., Science 254: 1497, 1991).Phosphorus-containing linkages include, but are not limited to,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates comprising 3′alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates comprising 3′-aminopho sphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′; see U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5, 177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563, 253; 5,571,799;5,587,361; and 5,625,050 (each of which is incorporated by reference).

Morpholino-based oligomeric compounds are described in Braasch et al.,Biochemistry 41(14):4503-4510, 2002; Genesis, volume 30, issue 3, 2001;Heasman, J., Dev. Biol., 243:209-214, 2002; Nasevicius et al., Nat.Genet. 26: 216-220, 2000; Lacerra et al., Proc. Natl. Acad. Sci. U.S.A.97:9591-9596, 2000; and U.S. Pat. No. 5,034,506. Cyclohexenyl nucleicacid oligonucleotide mimetics are described in Wang et al., J. Am. Chem.Soc. 122, 8595-8602, 2000.

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short-chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short-chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264, 562; 5, 264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596, 086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439 (each of which is herein incorporated by reference).

One or more substituted sugar moieties can also be included, e.g., oneof the following at the 2′ position: OH, SH, SCH₃, F, OCN, OCH₃ OCH₃,OCH₃ O(CH₂)n CH₃, O(CH₂)n NH₂ or O(CH₂)n CH₃, where n is from 1 to about10; Ci to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl,alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O—, S—, or N-alkyl; O—, S—,or N-alkenyl; SOCH3; SO2 CH3; ONO2; NO2; N3; NH2; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. A preferredmodification includes 2′-methoxyethoxy [2′-0—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl)] (Martin et al., Helv. Chim. Acta 78:486, 1995).Other preferred modifications include 2′-methoxy (2′-O-CH₃), 2′-propoxy(2′-OCH₂ CH₂CH₃) and 2′-fluoro (2′-F). Similar modifications may also bemade at other positions on the oligonucleotide, particularly the 3′position of the sugar on the 3′ terminal nucleotide and the 5′ positionof 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics, such as cyclobutyls in place of the pentofuranosyl group.

Inhibitory nucleic acids can also include, additionally oralternatively, nucleobase (often referred to in the art simply as“base”) modifications or substitutions. As used herein, “unmodified” or“natural” nucleobases include adenine (A), guanine (G), thymine (T),cytosine (C) and uracil (U). Modified nucleobases include nucleobasesfound only infrequently or transiently in natural nucleic acids, e.g.,hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly5-methylcytosine (also referred to as 5-methyl-2′ deoxycytosine andoften referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC),glycosyl HMC, and gentobiosyl HMC, as well as synthetic nucleobases,e.g., 2-aminoadenine, 2- (methylamino)adenine,2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or otherheterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine,5-bromouracil, 5- hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, and 2,6- diaminopurine. See Kornberg, A., DNAReplication, W. H. Freeman & Co., San Francisco, 1980, pp75-'7′7; andGebeyehu et al., Nucl. Acids Res. 15:4513, 1987. A “universal” baseknown in the art, e.g., inosine, can also be included. 5-Me-Csubstitutions have been shown to increase nucleic acid duplex stabilityby 0.6-1.2<0>C (Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., Eds.,Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp.276-278) and are presently preferred base substitutions.

It is not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligonucleotide or even atwithin a single nucleoside within an oligonucleotide.

In some embodiments, both a sugar and an internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, anoligonucleotide mimetic that has been shown to have excellenthybridization properties, is referred to as a peptide nucleic acid(PNA). In PNA compounds, the sugar-backbone of an oligonucleotide isreplaced with an amide containing backbone, for example, anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds comprise, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al, Science 254:1497-1500, 1991.

Inhibitory nucleic acids can also include one or more nucleobase (oftenreferred to in the art simply as “base”) modifications or substitutions.As used herein, “unmodified” or “natural” nucleobases comprise thepurine bases adenine (A) and guanine (G), and the pyrimidine basesthymine (T), cytosine (C), and uracil (U). Modified nucleobases compriseother synthetic and natural nucleobases, such as 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl, and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylquanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, and7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

Further, nucleobases comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in ‘The Concise Encyclopedia of PolymerScience And Engineering’, pages 858-859, Kroschwitz, J. I., Ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., AngewandleChemie, International Edition', 1991, 30, page 613, and those disclosedby Sanghvi, Y. S., Chapter 15, Antisense Research and Applications',pages 289- 302, Crooke, S. T. and Lebleu, B. ea., CRC Press, 1993.Certain of these nucleobases are particularly useful for increasing thebinding affinity of the oligomeric compounds of the invention. Theseinclude 5- substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and0-6 substituted purines, comprising 2-aminopropyladenine,5-propynyluracil, and 5- propynylcytosine. 5-methylcytosinesubstitutions have been shown to increase nucleic acid duplex stabilityby 0.6-1.2<0>C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., Eds,‘Antisense Research and Applications’, CRC Press, Boca Raton, 1993, pp.276-278) and are presently preferred base substitutions, even moreparticularly when combined with 2′-O-methoxyethyl sugar modifications.Modified nucleobases are described in U.S. Pat. No. 3,687,808, as wellas U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,750,692, and 5,681,941(each of which is herein incorporated by reference).

In some embodiments, the inhibitory nucleic acids are chemically linkedto one or more moieties or conjugates that enhance the activity,cellular distribution, or cellular uptake of the oligonucleotide. Suchmoieties comprise but are not limited to, lipid moieties such as acholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. U.S.A.86:6553-6556, 1989), cholic acid (Manoharan et al., Bioorg. Med. Chem.Lett. 4:1053-1060, 1994), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al, Ann. N. Y. Acad. Sci. 660:306-309, 1992; Manoharan etal., Bioorg. Med. Chem. Lett. 3:2765-2770, 1993), a thiocholesterol(Oberhauser et al., Nucl. Acids Res. 20, 533-538, 1992), an aliphaticchain, e.g., dodecandiol or undecyl residues (Kabanov et al., FEBS Lett.259:327-330, 1990; Svinarchuk et al., Biochimie 75:49- 54, 1993), aphospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl- rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett. 36:3651-3654, 1995; Shea et al., Nucl. AcidsRes.18:3777-3783, 1990), a polyamine or a polyethylene glycol chain(Mancharan et al., Nucleosides & Nucleotides 14:969-973, 1995), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett.36:3651-3654, 1995), a palmityl moiety (Mishra et al., Biochim. Biophys.Acta 1264: 229-237, 1995), or an octadecylamine or hexylamino-carbonyl-toxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther.277:923-937, 1996). See also U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552, 538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486, 603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762, 779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082, 830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5, 245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391, 723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5, 565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599, 928 and 5,688,941 (each of which is hereinincorporated by reference).

These moieties or conjugates can include conjugate groups covalentlybound to functional groups such as primary or secondary hydroxyl groups.Conjugate groups of the invention include intercalators, reportermolecules, polyamines, polyamides, polyethylene glycols, polyethers,groups that enhance the pharmacodynamic properties of oligomers, andgroups that enhance the pharmacokinetic properties of oligomers. Typicalconjugate groups include cholesterols, lipids, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance thepharmacodynamic properties, in the context of this invention, includegroups that improve uptake, enhance resistance to degradation, and/orstrengthen sequence-specific hybridization with the target nucleic acid.Groups that enhance the pharmacokinetic properties, in the context ofthis invention, include groups that improve uptake, distribution,metabolism, or excretion of the compounds of the present invention.Representative conjugate groups are disclosed in International PatentApplication No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No.6,287,860, which are incorporated herein by reference. Conjugatemoieties include, but are not limited to, lipid moieties such as acholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol,a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecylresidues, a phospholipid, e.g., di-hexadecyl-rac- glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, apolyamine or a polyethylene glycol chain, or adamantane acetic acid, apalmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941 (each of which is incorporated byreference).

The inhibitory nucleic acids useful in the present methods aresufficiently complementary to the target miRNA, i.e., hybridizesufficiently well and with sufficient specificity, to give the desiredeffect. “Complementary” refers to the capacity for pairing, throughhydrogen bonding, between two sequences comprising naturally ornon-naturally occurring bases or analogs thereof. For example, if a baseat one position of an inhibitory nucleic acid is capable of hydrogenbonding with a base at the corresponding position of a miRNA, then thebases are considered to be complementary to each other at that position.In some embodiments, 100% complementarity is not required. In someembodiments, 100% complementarity is required. Routine methods can beused to design an inhibitory nucleic acid that binds to the targetsequence with sufficient specificity.

While the specific sequences of certain exemplary target segments areset forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional target segments are readilyidentifiable by one having ordinary skill in the art in view of thisdisclosure. Target segments of 5, 6, 7, 8, 9, 10 or more nucleotides inlength comprising a stretch of at least five (5) consecutive nucleotideswithin the seed sequence, or immediately adjacent thereto, areconsidered to be suitable for targeting as well. In some embodiments,target segments can include sequences that comprise at least the 5consecutive nucleotides from the 5′-terminus of one of the seed sequence(the remaining nucleotides being a consecutive stretch of the same RNAbeginning immediately upstream of the 5′-terminus of the seed sequenceand continuing until the inhibitory nucleic acid contains about 5 toabout 30 nucleotides). In some embodiments, target segments arerepresented by RNA sequences that comprise at least the 5 consecutivenucleotides from the 3 ′-terminus of one of the seed sequence (theremaining nucleotides being a consecutive stretch of the same miRNAbeginning immediately downstream of the 3′-terminus of the targetsegment and continuing until the inhibitory nucleic acid contains about5 to about 30 nucleotides). One having skill in the art armed with thesequences provided herein will be able, without undue experimentation,to identify further preferred regions to target. In some embodiments, aninhibitory nucleic acid contain a sequence that is complementary to atleast 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 continguous nucleotides present in the target (e.g., thetarget miRNA, e.g., mature or precursor hsa-miR-155, or the targetmRNA).

Once one or more target regions, segments or sites have been identified,inhibitory nucleic acid compounds are chosen that are sufficientlycomplementary to the target, i.e., that hybridize sufficiently well andwith sufficient specificity (i.e., do not substantially bind to othernon-target RNAs), to give the desired effect.

In the context of this invention, hybridization means hydrogen bonding,which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogenbonding, between complementary nucleoside or nucleotide bases. Forexample, adenine and thymine are complementary nucleobases which pairthrough the formation of hydrogen bonds. Complementary, as used herein,refers to the capacity for precise pairing between two nucleotides. Forexample, if a nucleotide at a certain position of an oligonucleotide iscapable of hydrogen bonding with a nucleotide at the same position of amiRNA molecule or an mRNA molecule, then the inhibitory nucleic acid andthe miRNA or mRNA are considered to be complementary to each other atthat position. The inhibitory nucleic acids and the miRNA or mRNA arecomplementary to each other when a sufficient number of correspondingpositions in each molecule are occupied by nucleotides which canhydrogen bond with each other. Thus, “specifically hybridizable” and“complementary” are terms which are used to indicate a sufficient degreeof complementarity or precise pairing such that stable and specificbinding occurs between the inhibitory nucleic acid and the miRNA target.For example, if a base at one position of an inhibitory nucleic acid iscapable of hydrogen bonding with a base at the corresponding position ofa miRNA or a mRNA, then the bases are considered to be complementary toeach other at that position. 100% complementarity is not required.

It is understood in the art that a complementary nucleic acid sequenceneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. A complementary nucleic acid sequence forpurposes of the present methods is specifically hybridisable whenbinding of the sequence to the target miRNA or mRNA molecule interfereswith the normal function of the target miRNA or mRNA to cause a loss ofexpression or activity, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the sequence tonon-target RNA sequences under conditions in which specific binding isdesired, e.g., under physiological conditions in the case of in vivoassays or therapeutic treatment, and in the case of in vitro assays,under conditions in which the assays are performed under suitableconditions of stringency. For example, stringent salt concentration willordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate,preferably less than about 500 mM NaCl and 50 mM trisodium citrate, andmore preferably less than about 250 mM NaCl and 25 mM trisodium citrate.Low stringency hybridization can be obtained in the absence of organicsolvent, e.g., formamide, while high stringency hybridization can beobtained in the presence of at least about 35% formamide, and morepreferably at least about 50% formamide. Stringent temperatureconditions will ordinarily include temperatures of at least about 30°C., more preferably of at least about 37° C., and most preferably of atleast about 42° C. Varying additional parameters, such as hybridizationtime, the concentration of detergent, e.g., sodium dodecyl sulfate(SDS), and the inclusion or exclusion of carrier DNA, are well known tothose skilled in the art. Various levels of stringency are accomplishedby combining these various conditions as needed. In a preferredembodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mMtrisodium citrate, and 1% SDS. In a more preferred embodiment,hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodiumcitrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA(ssDNA). In a most preferred embodiment, hybridization will occur at 42°C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and200 μg/ml ssDNA. Useful variations on these conditions will be readilyapparent to those skilled in the art.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and even more preferably of at least about 68° C. Ina preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a more preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art. Hybridization techniques are well known to those skilled inthe art and are described, for example, in Benton and Davis (Science196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci. U.S.A.72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology,Wiley Interscience, N.Y., 2001); Berger and Kimmel (Guide to MolecularCloning Techniques, 1987, Academic Press, New York); and Sambrooke etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, New York.

In general, the inhibitory nucleic acids useful in the methods describedherein have at least 80% sequence complementarity to a target regionwithin the target nucleic acid, e.g., 90%, 95%, or 100% sequencecomplementarity to the target region within an miRNA. For example, anantisense compound in which 18 of 20 nucleobases of the antisenseoligonucleotide are complementary, and would therefore specificallyhybridize, to a target region would represent 90 percentcomplementarity. Percent complementarity of an inhibitory nucleic acidwith a region of a target nucleic acid can be determined routinely usingbasic local alignment search tools (BLAST programs) (Altschul et al., J.Mol. Biol. 215:403-410, 1990; Zhang and Madden, Genome Res. 7:649-656,1997). Antisense and other compounds of the invention that hybridize toan miRNA or a mRNA are identified through routine experimentation. Ingeneral the inhibitory nucleic acids must retain specificity for theirtarget, i.e., must not directly bind to, or directly significantlyaffect expression levels of, transcripts other than the intended target.

For further disclosure regarding inhibitory nucleic acids, please seeUS2010/0317718 (antisense oligos); US2010/0249052 (double-strandedribonucleic acid (dsRNA)); US2009/0181914 and USB2010/0234451 (LNAs);US2007/0191294 (siRNA analogues); US2008/0249039 (modified siRNA); andW02010/129746 and W02010/040112 (inhibitory nucleic acids).

Antisense

Antisense oligonucleotides are typically designed to block expression ofa DNA or RNA target by binding to the target and halting expression atthe level of transcription, translation, or splicing. Antisenseoligonucleotides of the present invention are complementary nucleic acidsequences designed to hybridize under stringent conditions to the targetmicroRNA or the target inflammatory marker mRNA. Thus, oligonucleotidesare chosen that are sufficiently complementary to the target, i.e., thathybridize sufficiently well and with sufficient specificity, to give thedesired effect.

Modified Bases/Locked Nucleic Acids (LNAs)

In some embodiments, the inhibitory nucleic acids used in the methodsdescribed herein comprise one or more modified bonds or bases. Modifiedbases include phosphorothioate, methylphosphonate, peptide nucleicacids, or locked nucleic acid (LNA) molecules. Preferably, the modifiednucleotides are locked nucleic acid molecules, including [alpha]-L-LNAs.LNAs comprise ribonucleic acid analogues wherein the ribose ring is“locked” by a methylene bridge between the 2′-oxgygen and the4′-carbon—i.e., oligonucleotides containing at least one LNA monomer,that is, one 2′-O,4′-C-methylene-β-D-ribofuranosyl nucleotide. LNA basesform standard Watson-Crick base pairs but the locked configurationincreases the rate and stability of the base pairing reaction (Jepsen etal., Oligonucleotides 14:130-146, 2004). LNAs also have increasedaffinity to base pair with RNA as compared to DNA. These propertiesrender LNAs especially useful as probes for fluorescence in situhybridization (FISH) and comparative genomic hybridization, as knockdowntools for miRNAs, and as antisense oligonucleotides to target mRNAs orother RNAs, e.g., miRNAs and mRNAs as described herein.

The LNA molecules can include molecules comprising 10-30, e.g., 12-24,e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 nucleotides in each strand, wherein one of the strands issubstantially identical, e.g., at least 80% (or more, e.g., 85%, 90%,95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatchednucleotide(s), to a target region in the miRNA or the mRNA. The LNAmolecules can be chemically synthesized using methods known in the art.

The LNA molecules can be designed using any method known in the art; anumber of algorithms are known, and are commercially available (e.g., onthe internet, for example at exiqon.com). See, e.g., You et al., Nuc.Acids. Res. 34:e60, 2006; McTigue et al., Biochemistry 43:5388-405,2004; and Levin et al., Nucl. Acids. Res. 34:e142, 2006. For example,“gene walk” methods, similar to those used to design antisense oligos,can be used to optimize the inhibitory activity of the LNA; for example,a series of oligonucleotides of 10-30 nucleotides spanning the length ofa target miRNA or mRNA can be prepared, followed by testing foractivity. Optionally, gaps, e.g., of 5-10 nucleotides or more, can beleft between the LNAs to reduce the number of oligonucleotidessynthesized and tested. GC content is preferably between about 30-60%.General guidelines for designing LNAs are known in the art; for example,LNA sequences will bind very tightly to other LNA sequences, so it ispreferable to avoid significant complementarity within an LNA.Contiguous runs of three or more Gs or Cs, or more than four LNAresidues, should be avoided where possible (for example, it may not bepossible with very short (e.g., about 9-10 nt) oligonucleotides). Insome embodiments, the LNAs are xylo-LNAs.

In some embodiments, the LNA molecules can be designed to target aspecific region of the miRNA. For example, a specific functional regioncan be targeted, e.g., a region comprising a seed sequence.Alternatively or in addition, highly conserved regions can be targeted,e.g., regions identified by aligning sequences from disparate speciessuch as primate (e.g., human) and rodent (e.g., mouse) and looking forregions with high degrees of identity. Percent identity can bedetermined routinely using basic local alignment search tools (BLASTprograms) (Altschul et al., J. Mol. Biol. 215:403-410, 1990; Zhang andMadden, Genome Res. 7:649-656, 1997), e.g., using the defaultparameters.

For additional information regarding LNAs see U.S. Pat. Nos. 6,268,490;6,734,291; 6,770,748; 6,794,499; 7,034,133; 7,053,207; 7,060,809;7,084,125; and 7,572,582; and U.S. Pre-Grant Pub. Nos. 2010/0267018;2010/0261175; and 2010/0035968; Koshkin et al., Tetrahedron54:3607-3630, 1998; Obika et al., Tetrahedron Lett. 39:5401-5404, 1998;Jepsen et al., Oligonucleotides 14:130-146 , 2004; Kauppinen et al.,Drug Disc. Today 2(3):287-290, 2005; and Ponting et al., Cell136(4):629-641, 2009, and references cited therein.

See also U.S. Ser. No. 61/412,862, which is incorporated by referenceherein in its entirety.

Antagomirs

In some embodiments, the antisense is an antagomir. Antagomirs arechemically-modified antisense oligonucleotides that target a microRNA(e.g., target hsa-miR-155). For example, an antagomir for use in themethods described herein can include a nucleotide sequence sufficientlycomplementary to hybridize to a miRNA target sequence of about 12 to 25nucleotides, preferably about 15 to 23 nucleotides.

In general, antagomirs include a cholesterol moiety, e.g., at the3′-end. In some embodiments, antagomirs have various modifications forRNase protection and pharmacologic properties such as enhanced tissueand cellular uptake. For example, in addition to the modificationsdiscussed above for antisense oligos, an antagomir can have one or moreof complete or partial 2′-O-methylation of sugar and/or aphosphorothioate backbone. Phosphorothioate modifications provideprotection against RNase activity and their lipophilicity contributes toenhanced tissue uptake. In some embodiments, the antagomir can includesix phosphorothioate backbone modifications; two phosphorothioates arelocated at the 5′-end and four at the 3′-end. See, e.g., Krutzfeldt etal., Nature 438:685-689, 2005; Czech, N. Engl. J. Med. 354:1194-1195,2006; Robertson et al., Silence 1:10, 2010; Marquez and McCaffrey, HumanGene Ther. 19(1):27-38, 2008; van Rooij et al., Circ. Res.103(9):919-928, 2008; and Liu et al., Int. J. Mol. Sci. 9:978-999, 2008.

Antagomirs useful in the present methods can also be modified withrespect to their length or otherwise the number of nucleotides making upthe antagomir. In general, the antagomirs are about 20-21 nucleotides inlength for optimal function, as this size matches the size of mostmature microRNAs. The antagomirs must retain specificity for theirtarget, i.e., must not directly bind to, or directly significantlyaffect expression levels of, transcripts other than the intended target.

In some embodiments, the inhibitory nucleic acid is locked and includesa cholesterol moiety (e.g., a locked antagomir).

siRNA

In some embodiments, the nucleic acid sequence that is complementary toa target miRNA or a target mRNA can be an interfering RNA, including butnot limited to a small interfering RNA (“siRNA”) or a small hairpin RNA(“shRNA”). Methods for constructing interfering RNAs are well known inthe art. For example, the interfering RNA can be assembled from twoseparate oligonucleotides, where one strand is the sense strand and theother is the antisense strand, wherein the antisense and sense strandsare self-complementary (i.e., each strand comprises nucleotide sequencethat is complementary to nucleotide sequence in the other strand; suchas where the antisense strand and sense strand form a duplex or doublestranded structure); the antisense strand comprises nucleotide sequencethat is complementary to a nucleotide sequence in a target nucleic acidmolecule or a portion thereof (i.e., an undesired gene) and the sensestrand comprises nucleotide sequence corresponding to the target nucleicacid sequence or a portion thereof. Alternatively, interfering RNA isassembled from a single oligonucleotide, where the self-complementarysense and antisense regions are linked by means of nucleic acid based ornon-nucleic acid-based linker(s). The interfering RNA can be apolynucleotide with a duplex, asymmetric duplex, hairpin or asymmetrichairpin secondary structure, having self-complementary sense andantisense regions, wherein the antisense region comprises a nucleotidesequence that is complementary to nucleotide sequence in a separatetarget nucleic acid molecule or a portion thereof and the sense regionhaving nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. The interfering can be a circularsingle-stranded polynucleotide having two or more loop structures and astem comprising self-complementary sense and antisense regions, whereinthe antisense region comprises nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof, and wherein thecircular polynucleotide can be processed either in vivo or in vitro togenerate an active siRNA molecule capable of mediating RNA interference.

In some embodiments, the interfering RNA coding region encodes aself-complementary RNA molecule having a sense region, an antisenseregion and a loop region. Such an RNA molecule when expressed desirablyforms a “hairpin” structure, and is referred to herein as an “shRNA.”The loop region is generally between about 2 and about 10 nucleotides inlength. In some embodiments, the loop region is from about 6 to about 9nucleotides in length. In some embodiments, the sense region and theantisense region are between about 15 and about 20 nucleotides inlength. Following post-transcriptional processing, the small hairpin RNAis converted into a siRNA by a cleavage event mediated by the enzymeDicer, which is a member of the RNase III family. The siRNA is thencapable of inhibiting the expression of a gene with which it shareshomology. For details, see Brummelkamp et al., Science 296:550-553,2002; Lee et al., Nature Biotechnol., 20, 500-505, 2002; Miyagishi andTaira, Nature Biotechnol. 20:497-500, 2002; Paddison et al., Genes &Dev. 16:948-958, 2002; Paul, Nature Biotechnol. 20, 505-508, 2002; Sui,Proc. Natl. Acad. Sci. U.S.A., 99(6):5515-5520, 2002; Yu et al., Proc.Natl. Acad. Sci. U.S.A. 99:6047-6052, 2002.

The target RNA cleavage reaction guided by siRNAs is highly sequencespecific. In general, siRNA containing a nucleotide sequences identicalto a portion of the target nucleic acid (i.e., a target regioncomprising the seed sequence of a target miRNA or mRNA) are preferredfor inhibition. However, 100% sequence identity between the siRNA andthe target gene is not required to practice the present invention. Thusthe invention has the advantage of being able to tolerate sequencevariations that might be expected due to genetic mutation, strainpolymorphism, or evolutionary divergence. For example, siRNA sequenceswith insertions, deletions, and single point mutations relative to thetarget sequence have also been found to be effective for inhibition.Alternatively, siRNA sequences with nucleotide analog substitutions orinsertions can be effective for inhibition. In general the siRNAs mustretain specificity for their target, i.e., must not directly bind to, ordirectly significantly affect expression levels of, transcripts otherthan the intended target.

Ribozymes

Trans-cleaving enzymatic nucleic acid molecules can also be used; theyhave shown promise as therapeutic agents for human disease (Usman &McSwiggen, Ann. Rep. Med. Chem. 30:285-294, 1995; Christoffersen andMarr, J. Med. Chem. 38:2023-2037, 1995). Enzymatic nucleic acidmolecules can be designed to cleave specific miRNA or mRNA targetswithin the background of cellular RNA. Such a cleavage event renders themiRNA or mRNA non-functional.

In general, enzymatic nucleic acids with RNA cleaving activity act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of an enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its activity. Afteran enzymatic nucleic acid has bound and cleaved its RNA target, it isreleased from that RNA to search for another target and can repeatedlybind and cleave new targets.

Several approaches such as in vitro selection (evolution) strategies(Orgel, Proc. R. Soc. London, B 205:435, 1979) have been used to evolvenew nucleic acid catalysts capable of catalyzing a variety of reactions,such as cleavage and ligation of phosphodiester linkages and amidelinkages, (Joyce, Gene, 82, 83-87, 1989; Beaudry et al., Science 257,635-641, 1992; Joyce, Scientific American 267, 90-97, 1992; Breaker etal., TIBTECH 12:268, 1994; Bartel et al., Science 261 :1411-1418, 1993;Szostak, TIBS 17, 89-93, 1993; Kumar et al., FASEB J., 9:1183, 1995;Breaker, Curr. Op. Biotech., 1:442, 1996). The development of ribozymesthat are optimal for catalytic activity would contribute significantlyto any strategy that employs RNA-cleaving ribozymes for the purpose ofregulating gene expression. The hammerhead ribozyme, for example,functions with a catalytic rate (kcat) of about 1 min⁻ in the presenceof saturating (10 rnM) concentrations of Mg²⁺ cofactor. An artificial“RNA ligase” ribozyme has been shown to catalyze the correspondingself-modification reaction with a rate of about 100 min⁻. In addition,it is known that certain modified hammerhead ribozymes that havesubstrate binding arms made of DNA catalyze RNA cleavage with multipleturn-over rates that approach 100 min⁻.

Sense Nucleic Acids

Agents useful in the methods of treatment described herein include sensenucleic acid molecules that increase the expression or activity of anyof the microRNAs (e.g., mature microRNA or precursor microRNA) listed inTables 2, 4, 6, 8, 10, 13, 15, 17, and 19, or increase the expression oractivity of any of the mRNAs encoding an inflammatory marker listed inTable 21. A sense nucleic acid can be contain a sequence that is atleast 80% (e.g., at least 90%, 95%, 96%, 97%, 98%, 99%, or 100%)identical to the sequence of any one of the microRNAs (e.g., maturemicroRNA or precursor microRNA) listed in Tables 2, 4, 6, 8, 10, 13, 15,17, and 19, or the sequence of any one of the mRNAs listed in Table 21.Sense nucleic acids can contain one or more of any of the modifications(e.g., backbone modifications, nucleobase modifications, sugarmodifications, or one or more conjugated molecules) described hereinwithout limitation. Methods of making and administering sense nucleicacids are known in the art. Additional methods of making and using sensenucleic acids are described herein.

Making and Using Inhibitory Nucleic Acids and Sense Nucleic Acids

The nucleic acid sequences used to practice the methods describedherein, whether RNA, cDNA, genomic DNA, vectors, viruses or hybridsthereof, can be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly.Recombinant nucleic acid sequences can be individually isolated orcloned and tested for a desired activity. Any recombinant expressionsystem can be used, including e.g., in vitro, bacterial, fungal,mammalian, yeast, insect, or plant cell expression systems.

Nucleic acid sequences of the invention (e.g., any of the inhibitorynucleic acids or sense nucleic acids described herein) can be insertedinto delivery vectors and expressed from transcription units within thevectors. The recombinant vectors can be DNA plasmids or viral vectors.Generation of the vector construct can be accomplished using anysuitable genetic engineering techniques well known in the art,including, without limitation, the standard techniques of PCR,oligonucleotide synthesis, restriction endonuclease digestion, ligation,transformation, plasmid purification, and DNA sequencing, for example asdescribed in Sambrook et al. Molecular Cloning: A Laboratory Manual.(1989)), Coffin et al. (Retroviruses. (1997)) and “RNA Viruses: APractical Approach” (Alan J. Cann, Ed., Oxford University Press,(2000)).

As will be apparent to one of ordinary skill in the art, a variety ofsuitable vectors are available for transferring nucleic acids of theinvention into cells. The selection of an appropriate vector to delivernucleic acids and optimization of the conditions for insertion of theselected expression vector into the cell, are within the scope of one ofordinary skill in the art without the need for undue experimentation.Viral vectors comprise a nucleotide sequence having sequences for theproduction of recombinant virus in a packaging cell. Viral vectorsexpressing nucleic acids of the invention can be constructed based onviral backbones including, but not limited to, a retrovirus, lentivirus,herpes virus, adenovirus, adeno-associated virus, pox virus, oralphavirus. The recombinant vectors (e.g., viral vectors) capable ofexpressing the nucleic acids of the invention can be delivered asdescribed herein, and persist in target cells (e.g., stabletransformants). For example, such recombinant vectors (e.g., arecombinant vector that results in the expression of an antisenseoligomer that is complementary to hsa-miR-155) can be administered into(e.g., injection or infusion into) the cerebrospinal fluid of thesubject (e.g., intracranial injection, intraparenchymal injection,intraventricular injection, and intrathecal injection, see, e.g., Bergenet al., Pharmaceutical Res. 25:983-998, 2007). A number of exemplaryrecombinant viral vectors that can be used to express any of the nucleicacids described herein are also described in Bergen et al. (supra).Additional examples of recombinant viral vectors are known in the art.

The nucleic acids provided herein (e.g., the inhibitory nucleic acids)can be further be complexed with one or more cationic polymers (e.g.,poly-L-lysine and poly(ethylenimine), cationic lipids (e.g.,1,2-dioleoyl-3-trimethylammonium propone (DOTAP),N-methyl-4-(dioleyl)methylpyridinium, and3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol), and/ornanoparticles (e.g., cationic polybutyl cyanoacrylate nanoparticles,silica nanoparticles, or polyethylene glycol-based nanoparticles) priorto administration to the subject (e.g., injection or infusion into thecerebrospinal fluid of the subject). Additional examples of cationicpolymers, cationic lipids, and nanoparticles for the therapeuticdelivery of nucleic acids are known in the art. The therapeutic deliveryof nucleic acids has also been shown to be achieved followingintrathecal injection of polyethyleneimine/DNA complexes (Wang et al.,Mol. Ther. 12:314-320, 2005). The methods for delivery of nucleic acidsdescribed herein are non-limiting. Additional methods for thetherapeutic delivery of nucleic acids to a subject are known in the art.

In some embodiments, the inhibitory nucleic acids (e.g., one or moreinhibitory nucleic acids targeting hsa-miR-155) can be administeredsystemically (e.g., intravenously, intaarterially, intramuscularly,subcutaneously, or intraperitoneally) or intrathecally (e.g., epiduraladministration). In some embodiments, the inhibitory nucleic acid isadministered in a composition (e.g., complexed with) one or morecationic lipids. Non-limiting examples of cationic lipids that can beused to administer one or more inhibitory nucleic acids (e.g., any ofthe inhibitory nucleic acids described herein) include: Lipofectamine,the cationic lipid molecules described in WO 97/045069, and U.S. PatentApplication Publication Nos. 2012/0021044, 2012/0015865, 2011/0305769,2011/0262527, 2011/0229581, 2010/0305198, 2010/0203112, and 2010/0104629(each of which is herein incorporated by reference).Nucleic acidsequences used to practice this invention can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g., Adams,J. Am. Chem. Soc. 105:661, 1983; Belousov, Nucleic Acids Res.25:3440-3444, 1997; Frenkel, Free Radic. Biol. Med. 19:373-380, 1995;Blommers, Biochemistry 33:7886-7896, 1994; Narang, Meth. Enzymol. 68:90,1994; Brown, Meth. Enzymol. 68:109, 1979; Beaucage, Tetra. Lett.22:1859, 1981; and U.S. Pat. No. 4,458,066.

Nucleic acid sequences of the invention can be stabilized againstnucleolytic degradation such as by the incorporation of a modification,e.g., a nucleotide modification. For example, nucleic acid sequences ofthe invention includes a phosphorothioate at least the first, second, orthird internucleotide linkage at the 5′ or 3′ end of the nucleotidesequence. As another example, the nucleic acid sequence can include a2′-modified nucleotide, e.g., a 2′-deoxy, 2′-deoxy-2′- fluoro,2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O--N-methylacetamido (2′-O--NMA). As another example, the nucleicacid sequence can include at least one 2′-O-methyl-modified nucleotide,and in some embodiments, all of the nucleotides include a 2′-O-methylmodification. In some embodiments, the nucleic acids are “locked,” i.e.,comprise nucleic acid analogues in which the ribose ring is “locked” bya methylene bridge connecting the 2′-O atom and the 4′-C atom (see,e.g., Kaupinnen et al., Drug Disc. Today 2(3):287-290, 2005; Koshkin etal., J. Am. Chem. Soc., 120(50):13252-13253, 1998). For additionalmodifications see US 2010/0004320, US 2009/0298916, and US 2009/0143326(each of which is incorporated by reference).

Techniques for the manipulation of nucleic acids used to practice thisinvention, such as, e.g., subcloning, labeling probes (e.g.,random-primer labeling using Klenow polymerase, nick translation,amplification), sequencing, hybridization, and the like are welldescribed in the scientific and patent literature, see, e.g., Sambrooket al., Molecular Cloning; A Laboratory Manual 3d ed. (2001); CurrentProtocols in Molecular Biology, Ausubel et al., Eds. (John Wiley & Sons,Inc., N.Y. 2010); Kriegler, Gene Transfer and Expression: A LaboratoryManual (1990); Laboratory Techniques In Biochemistry And MolecularBiology: Hybridization With Nucleic Acid Probes, Part I. Theory andNucleic Acid Preparation, Tijssen, Ed. Elsevier, N.Y. (1993).

Antibodies and Recombinant Proteins

One or more antibodies that specifically bind to a protein encoded byany of the inflammatory marker genes listed in Table 21 can also beadministered to a subject to treat a neurodegenerative disease.Antibodies that specifically bind to a protein listed in Table 21 areeither commercially available or can be generated using standard methodsknown in the art. For example, a polyclonal antibody that specificallybinds to a protein listed in Table 21 can be generated by immunizing amammal with the purified protein and isolating antibodies from themammal that specifically bind to the purified protein. The antibodiesused can be a monoclonal or polyclonal antibody. The antibodiesadministered can be a immunoglobulin G or immunoglobulin M. Theantibodies administered can be chimeric (e.g., a humanized antibody) ora human antibody. The antibodies used can also be an antibody fragment(e.g., a Fab, F(ab')₂, Fv, and single chain Fv (scFv) fragment).

One or more inflammatory marker proteins listed in Table 20 can also beadministered to a subject for the treatment of a neurodegenerativedisorder. Several methods are known in the art for the production of arecombinant protein using molecular biology and cell culture techniques.For example, an inflammatory marker protein encoded by a mRNA sequencelisted in Table 20 can be transfected into a bacterial, yeast, ormammalian cell (using a protein expression plasmid or viral vector) thatallows for the expression of the inflammatory by the transfected cell.The transfected cells or the culture medium can be collected, and therecombinant inflammatory marker protein purified using methods known inthe art. The inflammatory marker proteins administered to the subjectcan contain a sequence having at least 80% (e.g., at least 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100%) identity to any of the amino acidsequences listed in Table 20. The inflammatory marker proteinsadministered to the subject can further contain a modification (e.g., apolyethylene glycol or an HIV tat protein, or any other moiety thatincreases the cellular permeability of the inflammatory marker protein).

Pharmaceutical Compositions

The methods described herein can include the administration ofpharmaceutical compositions and formulations comprising any one or more(e.g., two, three, four, or five) of the inhibitory nucleic acids (e.g.,one or more inhibitory nucleic acids targeting hsa-miR-155), sensenucleic acids, inflammatory marker proteins, or antibodies describedherein.

In some embodiments, the compositions are formulated with apharmaceutically acceptable carrier. The pharmaceutical compositions andformulations can be administered parenterally, topically, orally or bylocal administration, such as by aerosol or transdermally. Thepharmaceutical compositions can be formulated in any way and can beadministered in a variety of unit dosage forms depending upon thecondition or disease and the degree of illness, the general medicalcondition of each patient, the resulting preferred method ofadministration and the like. Details on techniques for formulation andadministration of pharmaceuticals are well described in the scientificand patent literature, see, e.g., Remington: The Science and Practice ofPharmacy, 21st ed., 2005.

The inhibitory nucleic acids can be administered alone or as a componentof a pharmaceutical formulation (composition). The compounds may beformulated for administration, in any convenient way for use in human orveterinary medicine. Wetting agents, emulsifiers and lubricants, such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, release agents, coating agents, sweetening, flavoring andperfuming agents, preservatives, and antioxidants can also be present inthe compositions. In some embodiments, one or more cationic lipids,cationic polymers, or nanoparticles can be included in compositionscontaining the one or more inhibitory nucleic acids (e.g., compositionscontaining one or more inhibitory nucleic acids targeting hsa-miR-155).

Formulations of the compositions of the invention include those suitablefor intradermal, inhalation, oral/ nasal, topical, parenteral, rectal,and/or intravaginal administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient (e.g.,nucleic acid sequences of this invention) which can be combined with acarrier material to produce a single dosage form will vary dependingupon the host being treated, the particular mode of administration,e.g., intradermal or inhalation. The amount of active ingredient whichcan be combined with a carrier material to produce a single dosage formwill generally be that amount of the compound which produces atherapeutic effect.

Pharmaceutical formulations of this invention can be prepared accordingto any method known to the art for the manufacture of pharmaceuticals.Such drugs can contain sweetening agents, flavoring agents, coloringagents, and preserving agents. A formulation can be admixtured withnontoxic pharmaceutically acceptable excipients which are suitable formanufacture. Formulations may comprise one or more diluents,emulsifiers, preservatives, buffers, excipients, etc., and may beprovided in such forms as liquids, powders, emulsions, lyophilizedpowders, sprays, creams, lotions, controlled release formulations,tablets, pills, gels, on patches, in implants, etc.

Pharmaceutical formulations for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art inappropriate and suitable dosages. Such carriers enable thepharmaceuticals to be formulated in unit dosage forms as tablets, pills,powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries,suspensions, etc., suitable for ingestion by the patient. Pharmaceuticalpreparations for oral use can be formulated as a solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable additional compounds, if desired, toobtain tablets or dragee cores. Suitable solid excipients arecarbohydrate or protein fillers include, e.g., sugars, includinglactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,potato, or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; andgums including arabic and tragacanth; and proteins, e.g., gelatin andcollagen. Disintegrating or solubilizing agents may be added, such asthe cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate. Push-fit capsules can contain activeagents mixed with a filler or binders such as lactose or starches,lubricants such as talc or magnesium stearate, and, optionally,stabilizers. In soft capsules, the active agents can be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycol with or without stabilizers.

Aqueous suspensions can contain an active agent (e.g., inhibitorynucleic acids or sense nucleic acids described herein) in admixture withexcipients suitable for the manufacture of aqueous suspensions, e.g.,for aqueous intradermal injections. Such excipients include a suspendingagent, such as sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth, and gum acacia, and dispersing or wetting agents such as anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long-chain aliphaticalcohol (e.g., heptadecaethylene oxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensationproduct of ethylene oxide with a partial ester derived from fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate).The aqueous suspension can also contain one or more preservatives suchas ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents, and one or more sweetening agents, such assucrose, aspartame, or saccharin. Formulations can be adjusted forosmolarity.

In some embodiments, oil-based pharmaceuticals are used foradministration of nucleic acid sequences of the invention. Oil-basedsuspensions can be formulated by suspending an active agent in avegetable oil, such as arachis oil, olive oil, sesame oil, or coconutoil, or in a mineral oil such as liquid paraffin; or a mixture of these.See e.g., U.S. Pat. No. 5,716,928, describing using essential oils oressential oil components for increasing bioavailability and reducinginter- and intra-individual variability of orally administeredhydrophobic pharmaceutical compounds (see also U.S. Pat. No. 5,858,401).The oil suspensions can contain a thickening agent, such as beeswax,hard paraffin, or cetyl alcohol. Sweetening agents can be added toprovide a palatable oral preparation, such as glycerol, sorbitol, orsucrose. These formulations can be preserved by the addition of anantioxidant such as ascorbic acid. As an example of an injectable oilvehicle, see Minto, J. Pharmacol. Exp. Ther. 281:93-102, 1997.

Pharmaceutical formulations can also be in the form of oil-in-wateremulsions. The oily phase can be a vegetable oil or a mineral oil,described above, or a mixture of these. Suitable emulsifying agentsinclude naturally-occurring gums, such as gum acacia and gum tragacanth,naturally occurring phosphatides, such as soybean lecithin, esters, orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan mono-oleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. Theemulsion can also contain sweetening agents and flavoring agents, as inthe formulation of syrups and elixirs. Such formulations can alsocontain a demulcent, a preservative, or a coloring agent. In alternativeembodiments, these injectable oil-in-water emulsions of the inventioncomprise a paraffin oil, a sorbitan monooleate, an ethoxylated sorbitanmonooleate, and/or an ethoxylated sorbitan trioleate.

The pharmaceutical compounds can also be administered by in intranasal,intraocular and intravaginal routes including suppositories,insufflation, powders and aerosol formulations (for examples of steroidinhalants, see e.g., Rohatagi, J. Clin. Pharmacol. 35:1187-1193, 1995;Tjwa, Ann. Allergy Asthma Immunol. 75:107-111, 1995). Suppositoriesformulations can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at body temperatures and will therefore melt in the body torelease the drug. Such materials are cocoa butter and polyethyleneglycols.

In some embodiments, the pharmaceutical compounds can be deliveredtransdermally, by a topical route, formulated as applicator sticks,solutions, suspensions, emulsions, gels, creams, ointments, pastes,jellies, paints, powders, and aerosols.

In some embodiments, the pharmaceutical compounds can also be deliveredas microspheres for slow release in the body. For example, microspherescan be administered via intradermal injection of drug which slowlyrelease subcutaneously; see Rao, J. Biomater Sci. Polym. Ed. 7:623-645,1995; as biodegradable and injectable gel formulations, see, e.g., Gao,Pharm. Res. 12:857-863, 1995; or, as microspheres for oraladministration, see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997.

In some embodiments, the pharmaceutical compounds can be parenterallyadministered, such as by intravenous (IV) administration oradministration into a body cavity, a lumen of an organ, or into thecranium (e.g., intracranial injection or infusion) or the cerebrospinalfluid of a subject. These formulations can comprise a solution of activeagent dissolved in a pharmaceutically acceptable carrier. Acceptablevehicles and solvents that can be employed are water and Ringer'ssolution, an isotonic sodium chloride. In addition, sterile fixed oilscan be employed as a solvent or suspending medium. For this purpose anybland fixed oil can be employed including synthetic mono- ordiglycerides. In addition, fatty acids, such as oleic acid can likewisebe used in the preparation of injectables. These solutions are sterileand generally free of undesirable matter. These formulations may besterilized by conventional, well known sterilization techniques. Theformulations may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents, e.g.,sodium acetate, sodium chloride, potassium chloride, calcium chloride,sodium lactate, and the like. The concentration of active agent in theseformulations can vary widely, and will be selected primarily based onfluid volumes, viscosities, body weight, and the like, in accordancewith the particular mode of administration selected and the patient'sneeds. For IV administration, the formulation can be a sterileinjectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension can be formulated using thosesuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation can also be a suspension in a nontoxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol. The administration can be by bolus or continuousinfusion (e.g., substantially uninterrupted introduction into a bloodvessel for a specified period of time).

In some embodiments, the pharmaceutical compounds and formulations canbe lyophilized. Stable lyophilized formulations comprising an inhibitorynucleic acid or a sense nucleic acid can be made by lyophilizing asolution comprising a pharmaceutical of the invention and a bulkingagent, e.g., mannitol, trehalose, raffinose, and sucrose, or mixturesthereof. A process for preparing a stable lyophilized formulation caninclude lyophilizing a solution about 2.5 mg/mL protein, about 15 mg/mLsucrose, about 19 mg/mL NaCl, and a sodium citrate buffer having a pHgreater than 5.5, but less than 6.5. See, e.g., US2004/0028670.

The compositions and formulations can be delivered by the use ofliposomes. By using liposomes, particularly where the liposome surfacecarries ligands specific for target cells, or are otherwisepreferentially directed to a specific organ, one can focus the deliveryof the active agent into target cells in vivo. See, e.g., U.S. Pat. Nos.6,063,400; 6,007,839; Al-Muhammed, J. Microencapsul. 13:293-306, 1996;Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp.Pharm. 46:1576-1587, 1989.

The formulations of the invention can be administered for prophylacticand/or therapeutic treatments. In some embodiments, for therapeuticapplications, compositions are administered to a subject who is at riskof or has a disorder described herein, in an amount sufficient to cure,alleviate or partially arrest the clinical manifestations of thedisorder or its complications; this can be called a therapeuticallyeffective amount. For example, in some embodiments, pharmaceuticalcompositions of the invention are administered in an amount sufficientto reduce the number of symptoms or reduce the severity, duration, orfrequency of one or more symptoms of a neurodegenerative disorder in asubject.

The amount of pharmaceutical composition adequate to accomplish this isa therapeutically effective dose. The dosage schedule and amountseffective for this use, i.e., the dosing regimen, will depend upon avariety of factors, including the stage of the disease or condition, theseverity of the disease or condition, the general state of the patient'shealth, the patient's physical status, age, and the like. In calculatingthe dosage regimen for a patient, the mode of administration also istaken into consideration.

The dosage regimen also takes into consideration pharmacokineticsparameters well known in the art, i.e., the active agents' rate ofabsorption, bioavailability, metabolism, clearance, and the like (see,e.g., Hidalgo-Aragones, J. Steroid Biochem. Mol. Biol. 58:611-617, 1996;Groning, Pharmazie 51:337-341, 1996; Fotherby, Contraception 54:59-69,1996; Johnson, i J. Pharm. Sci. 84:1144-1146, 1995; Rohatagi, Pharmazie50:610-613, 1995; Brophy, Eur. J. Clin. Pharmacol. 24:103-108, 1983;Remington: The Science and Practice of Pharmacy, 21st ed., 2005). Thestate of the art allows the clinician to determine the dosage regimenfor each individual patient, active agent, and disease or conditiontreated. Guidelines provided for similar compositions used aspharmaceuticals can be used as guidance to determine the dosageregiment, i.e., dose schedule and dosage levels, administered practicingthe methods of the invention are correct and appropriate.

Single or multiple administrations of formulations can be givendepending on for example: the dosage and frequency as required andtolerated by the patient, and the like. The formulations should providea sufficient quantity of active agent to effectively treat, prevent orameliorate conditions, diseases, or symptoms.

In alternative embodiments, pharmaceutical formulations for oraladministration are in a daily amount of between about 1 to 100 or moremg per kilogram of body weight per day. Lower dosages can be used, incontrast to administration orally, into the blood stream, into a bodycavity or into a lumen of an organ. Substantially higher dosages can beused in topical or oral administration or administering by powders,spray, or inhalation. Actual methods for preparing parenterally ornon-parenterally administrable formulations will be known or apparent tothose skilled in the art and are described in more detail in suchpublications as Remington: The Science and Practice of Pharmacy, 21sted., 2005.

Various studies have reported successful mammalian dosing usingcomplementary nucleic acid sequences. For example, Esau C., et al., CellMetabolism, 3(2):87-98, 2006, reported dosing of normal mice withintraperitoneal doses of miR-122 antisense oligonucleotide ranging from12.5 to 75 mg/kg twice weekly for 4 weeks. The mice appeared healthy andnormal at the end of treatment, with no loss of body weight or reducedfood intake. Plasma transaminase levels were in the normal range (AST³/₄ 45, ALT ³/₄35) for all doses with the exception of the 75 mg/kg doseof miR-122 ASO, which showed a very mild increase in ALT and AST levels.They concluded that 50mg/kg was an effective, non-toxic dose. Anotherstudy by Krutzfeldt J., et al., Nature 438, 685-689, 2005, injectedanatgomirs to silence miR-122 in mice using a total dose of 80, 160 or240 mg per kg body weight. The highest dose resulted in a complete lossof miR-122 signal. In yet another study, locked nucleic acids (“LNAs”)were successfully applied in primates to silence miR-122. Elmen et al.,Nature 452, 896-899, 2008, report that efficient silencing of miR-122was achieved in primates by three doses of 10 mg kg−1 LNA-antimiR,leading to a long-lasting and reversible decrease in total plasmacholesterol without any evidence for LNA-associated toxicities orhistopathological changes in the study animals.

In some embodiments, the methods described herein can includeco-administration with other drugs or pharmaceuticals, e.g., any of thetreatments of a neurodegenerative disorder described herein.

Kits

Also provided herein are kits containing one or more (e.g., at least 2,3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20) of any of the probes,inhibitory nucleic acids, sense nucleic acids, inflammatory markerproteins, or antibodies described herein (in any combination). In someembodiments, the kits can include instructions for performing any of themethods described herein.

In some embodiments, the kit can contain at least two primers (e.g., atleast 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 36, 38,or 40) for amplifying a sequence present within any of the microRNAslisted in Tables 1-19 (e.g., mature microRNA or precursor microRNA) orfor amplifying a sequence present within any of the mRNAs listed inTables 20 and 21.

In some embodiments, the kits contain two or more sets of primer (e.g.,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, or 109pairs of primers) that amplify a sequence present within one or more(e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,or 109 pairs of primers) of the microRNAs listed in any one of Tables1-11 (e.g., one or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, or 109) of the microRNAs from Tables 1 and 2;Tables 3 and 4; Tables 5 and 6; Tables 7 and 8; Tables 9 and 10; andTable 11) and/or that amplify a sequence present within one or more(e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, or 95) of the mRNAs listed in Table 20 and/or Table 21 (e.g.,ALS diagnostic kits).

In some embodiments, the kits contain two or more sets of primer (e.g.,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, or 109pairs of primers) that amplify a sequence present within one or more(e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,or 109 pairs of primers) of the microRNAs listed in any one of Tables1-11 (e.g., one or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, or 109) of the microRNAs from Tables 1 and 2;Tables 3 and 4; Tables 5 and 6; Tables 7 and 8; Tables 9 and 10; andTable 11) and/or that amplify a sequence present within one or more(e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, or 95) of the mRNAs listed in Table 20 and/or Table 21 (e.g.,ALS diagnostic kits).

In some embodiments, the kits contain two or more antisenseoligonucleotides (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, or 109 pairs of primers) that collectively arecapable of hybridizing to one or (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, or 109) of the microRNAs listed inany one of Tables 1, 2, and12-19 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, or 109 microRNAs from Tables 1 and 2;Tables 12 and 13; Tables 14 and 15; Tables 16 and 17; and Tables 18 and19) and/or that are collectively capable of hybridizing to one or more(e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, or 95) of the mRNAs listed in Table 20 and/or Table 21 (e.g., MSdiagnostic kits).

In some embodiments, the kits contain two or more antisenseoligonucleotides (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, or 109 antisense oligonucleotides) that collectivelyare capable of hybridizing to one or more (e.g., 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, or 109) of the microRNAslisted in any one of Tables 1, 2, and12-19 (e.g., 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, or 109 microRNAs fromTables 1 and 2; Tables 12 and 13; Tables 14 and 15; Tables 16 and 17;and Tables 18 and 19) and/or that collectively are capable ofhybridizing to one or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, or 95) of the mRNAs listed in Table20 and/or Table 21 (e.g., MS diagnostic kits).

In some embodiments, the kit can contain at least two antisensemolecules (e.g., at least 4, 6, 8, 10, 12, 14, 16, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, or 40) for hybridizing with a sequence presentwithin any of the microRNAs listed in Tables 1-19 (e.g., mature microRNAor precursor microRNA) or a sequence within any of the mRNAs listed inTables 20 and 21.

In some embodiments, the kits can contain at least one inhibitorynucleic acid and/or at least one sense nucleic acid (e.g., any of theinhibitory nucleic acids or sense nucleic acids described herein). Insome embodiments, the kit contains at least one inhibitory nucleic acid(e.g., at least one inhibitory nucleic acid targeting hsa-miR-155)formulated for intrathecal or intracranial injection or infusion.

In some embodiments, the kits can contain at least one (e.g., at leasttwo, three, four, five, or six) antibody that specifically binds to anyone of the proteins encoded by any of the inflammatory marker geneslisted in Table 20 or Table 21 (e.g., any of the variety of antibodiesor antibody fragments described herein). In some embodiments, theantibodies can be labeled (e.g., labeled with a fluorophore, aradioisotope, an enzyme, biotin, or avidin).

In some embodiments, the kit further contains at least one additionaltherapeutic agent (e.g., one or more of KNS760704, SB509, ceftriaxone,minocycline, rilutek, and riluzole). In some embodiments, the kitfurther contains instructions for administering the at least one agent(e.g., one or more inhibitory nucleic acids) to a subject having ordiagnosed as having a neurodegenerative disease (e.g., sporadic ALSand/or familial ALS, or MS).

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

EXAMPLES Example 1. MicroRNA Deregulation in Ly6C^(Hi)Monocytes,Ly6C^(Low)Monocytes, and CD39⁺ Microglia in a Mouse Model of ALS(SOD1^(G93A) Mice)

Lys6C^(H1)/CCR2⁺monocytes participate in tissue damage and diseasepathogenesis in a variety of conditions, including an animal model of MS(King et al., Blood 113:3190-3197, 2009). Experiments were to performedto compare the microRNA expression profile in CD39 ⁺ microglia (FIG.1A-1C), Ly6C^(Hi) monocytes (FIG. 2A-2C), and Ly6C^(L)″' monocytes (FIG.3A-3C), and in the mouse SOD1^(G93A) model of ALS at the presymptomaticstage (60 days), at the time of onset of symptoms, and at the end stageof disease to the expression in the same cells in non-transgeniclitermates.

These data were gathered using rodent TaqMan Low Density Arrays (TaqManMicroRNA Assays containing 364 mouse microRNA assays (n=2 arrays foreach group; pool of 5-6 mice per group). The microarray data werenormalized using quantile (R software) to remove variation betweensamples. MicroRNA expression level was normalized using dCT against U6miRNA (internal control) and geometric mean across all samples. Afterthe normalization step, analysis of variants between groups (ANOVA) wasused to define significantly altered microRNAs across all disease stagesin SOD1 mice (using a false discovery rate of ≤0.1).

MicroRNA profiling of spleen-derived Ly6C^(Hi) and Ly6C^(Low) monocytesof the SOD1 mice during all stages of the disease showed 32significantly dysregulated microRNAs in Ly6C^(Hi) splenic monocytes and23 dysregulated microRNAs in Ly6C^(Low) splenic monocytes. All of thedysregulated microRNAs in the monocyte subsets were observed one monthprior to clinical onset and during disease progression. The majority ofthese microRNAs were not overlapping between Lys6C^(Hi) monocytes andLy6C^(Low) monocytes, suggesting that these different monocyte subsetshave different functions during disease progression.Inflammation-related microRNAs such as let-7a, miR-27a, miR-34a,miR-132, miR-146a, miR-451, and miR-155 were significantly upregulatedin the Ly6C^(Hi) monocytes in the spleen during disease progression inthe SOD1 mice (FIG. 2). Ingenuity pathway analysis of the microRNAprofile of Ly6C^(Hi) monocytes in SOD1 mice identified patterns ofmicroRNA expression observed in primary muscular disorders (FIG. 4).

The data from microRNA expression profiling of CD39⁺ microglia in theSOD1 mouse show that 24 microRNAs were upregulated and two microRNAswere downregulated compared to the same cells in non-transgeniclitermates. These microRNAs were different from the microRNAsdysregulated in Ly6C^(Hi) monocytes, with the exception of 6 microRNAs(let-7a, miR-27a, miR-34a, miR-132, miR-146a, and miR-155). These datademonstrate differences between resident microglia identified by CD39and infiltrating Ly6C monocytes, and identify a unique microRNA patternin microglia in SOD1 mice.

Example 2. MicroRNAs dysregulated in CD14⁺CD16⁻ monocytes in subjectswith ALS an MS

In view of the unique microRNA profiles observed in mouse monocytes,microRNA Expression Profiling was performed on human blood-derivedCD14⁺CD16⁻ monocytes (Ly6C^(Hi) nalog) from ALS subjects and MSsubjects. In these experiments, nCounter expression profiling of 664microRNAs in blood-derived CD14⁺CD16⁻ monocytes from subjects havingsporadic ALS (n=8), subjects having relapsing-remitting MS (n=8), andhealthy controls (n=8). The microRNA expression level was normalizedagainst a geometric mean of five house- keeping genes (ACTB, B2M, GAPDH,RPL19, and RPLPO). A heatmap comparing the microRNA expression inmonocytes from ALS or MS subjects compared to the expression in healthycontrols was generated using ANOVA with Dunnett's post hoc test (p<0.01)(FIGS. 5, 6, and 33, respectively). FIG. 7A depicts the number ofmicroRNAs uniquely upregulated in CD14⁺CD16⁻ monocytes from ALS and MSsubjects, as well as the number of microRNAs upregulated in CD14⁺CD16⁻monocytes from both ALS and MS subjects. FIG. 7A also depicts the numberof microRNAs uniquely downregulated in CD14⁺CD16⁻ monocytes from ALS andMS subjects, as well as the number of microRNAs downregulated inCD14⁺CD16⁻ monocytes from both ALS and MS subjects. FIG. 7B is a volcanoplot showing the significantly deregulated microRNAs in CD14⁺CD16⁻monocytes from ALS subjects compared to the expression of the microRNAsin CD14⁺CD16⁻ monocytes from healthy controls. FIG. 7C is a volcano plotshowing the significantly deregulated microRNAs in CD14⁺CD16⁻ monocytesfrom MS subjects compared to the expression of the microRNAs inCD14⁺CD16⁻ monocytes from healthy controls. FIG. 8 provides a summary ofthe microRNAs deregulated in CD14⁺CD16⁻ monocytes from ALS and MSsubjects compared to the expression of the microRNAs in CD14⁺CD16⁻monocytes from healthy controls.

The dysregulation of specific microRNAs in CD14⁺CD16⁻ monocytes from ALSand MS subjects (as compared to healthy controls) were confirmed usingreal-time PCR. For example, real-time PCR was used to confirm theupregulation of hsa-miR-27a, hsa-miR-190, hsa-miR- 500, hsa-miR-155, andhsa-miR-532-3p in CD14⁺CD16⁻ monocytes from ALS subjects (n=11) comparedto the expression of these microRNAs in CD14⁺CD16⁻ monocytes fromhealthy controls (n=8) (two-tiled Mann-Whitney t-test) (FIG. 9).Additional real-time PCR experiments were performed to confirm theunique upregulation of microRNAs in CD14⁺CD16⁻ monocytes from ALSsubjects (n=8; clinical scoring of these subjects is shown in FIGS. 10Aand 10B) as compared to the expression of these microRNAs in CD14⁺CD16⁻monocytes from both MS subjects and healthy controls (FIG. 10C). Thedata in FIG. 10C show that 20 different microRNAs are uniquelyupregulated in CD14⁺CD16⁻ monocytes as compared the expression of thesemicroRNAs in CD14⁺CD16⁻ monocytes from MS subjects and healthy controls:hsa-miR-19b, hsa-miR-106b, hsa-miR-30b, hsa-miR-21, hsa-miR-142-5p,hsa-miR-27a, hsa-miR-16, hsa-miR-374a, hsa-miR-374b, hsa-miR-101,hsa-miR-340, hsa-miR-30e, hsa-miR-29c, hsa-miR-29a, hsa-miR-223,hsa-miR-26a, hsa-miR-26b, hsa-miR-24, hsa-miR-181a, and hsa-miR-103. Theunique upregulation of hsa-miR-27a, hsa-miR-155, hsa-miR-146a, andhsa-miR-532-3p in CD14⁺CD16⁻ monocytes from ALS subjects as compared tothe expression of these microRNAs in CD14⁺CD16⁻ monocytes from MSsubjects and healthy controls was also confirmed in a second set ofreal-time PCR experiments (FIG. 11) (microRNA expression was normalizedagainst dCT using U6 miRNA). Additional real-time PCR experiments wereperformed to confirm the unique down-regulation of hsa-miR-518f,hsa-miR-206, hsa-miR-204, hsa-miR-137, hsa-miR-453, hsa-miR-603,hsa-miR-1297, hsa-miR-192, hsa-miR-526a, hsa-miR- 615-5p, hsa-miR-655,hsa-miR-450b-5p, hsa-miR-548b-3p, hsa-miR-584, hsa-miR-548f, hsa-miR-300, hsa-miR-302c, hsa-miR-328, hsa-miR-421, and hsa-miR-580 inCD14⁺CD16⁻ monocytes from ALS subjects compared to the expression ofthese microRNAs in CD14⁺CD16⁻ monocytes from both MS subjects andhealthy controls (FIG. 12).

A further set of real-time PCR experiments were performed to verify theupregulation of hsa-miR-24, hsa-miR-93, hsa-miR-20a, hsa-let-7a,hsa-miR-30c, hsa-miR-181a, hsa-miR-432- 3p, and hsa-miR-1260 inCD14⁺CD16⁻ monocytes from both ALS and MS subjects as compared to theexpression of these microRNAs in CD14⁺CD16⁻ monocytes from healthycontrols (FIG. 13). An additional set of real-time PCR experiments wereperformed to verify the upregulation of hsa-miR-320c, hsa-miR-27b,hsa-miR-664, hsa-miR-423-5p, and hsa-miR-92a in CD14⁺CD16⁻ monocytesfrom MS subjects as compared to healthy controls (FIG. 14). These dataalso show confirm that hsa-miR-664 is uniquely upregulated in CD14⁺CD16⁻monocytes from MS subjects as compared the expression of this microRNAin CD14⁺CD16⁻ monocytes from both ALS subjects and healthy controls(FIG. 14).

In addition, the unique downregulation of hsa-miR-142-3p, hsa-miR-15a,hsa-miR-1537, hsa-miR-362-3p, and hsa-miR-148b in CD14⁺CD16⁻ monocytesfrom MS subjects compared to the expression of these microRNAs inCD14⁺CD16⁻ monocytes from ALS subjects and healthy controls wasconfirmed using real-time PCR (FIG. 15).

Example 3. Abnormal MicroRNA Levels in Cerebrospinal Fluid from Subjectshaving Sporadic ALS and Familial ALS

MicroRNA expression profiling was also performed using cerebrospinalfluid (CSF) from subjects having sporadic ALS and familial ALS. Thelevels of microRNAs in the CSF from both sporadic ALS (n=10) andfamilial ALS (n=5) subjects was compared to the levels of microRNAs inthe CSF of healthy controls (n=10). The resulting data show thathsa-miR-27b is increased in the CSF of subjects having both sporadic andfamilial ALS as compared to the level of this microRNA in the CSF ofhealthy controls, and that hsa-miR-99b, hsa-miR-146a, hsa-miR-150,hsa-miR-328, and hsa-miR-532-3p are uniquely upregulated in CSF fromsubjects having sporadic ALS compared to the levels of these microRNAsin CSF from healthy controls or subjects having familial ALS (FIG. 16).

Example 4. Inflammation-Related Genes are also dysregulated inCD14⁺CD16⁻ monocytes from subjects having ALS and MS

Expression profiling analysis of 179 inflammation-related genes(“inflammatory marker genes”) was performed using CD14⁺CD16⁻ monocytesfrom ALS subjects (n=8), MS subjects (n=11), and healthy controls(n=10). A heatmap showing the change in expression of differentinflammatory marker genes in CD14⁺CD16⁻ monocytes from ALS or MSsubjects, as compared to the expression of these genes in CD14⁺CD16⁻monocytes from healthy subjects is shown in FIG. 17). A volcano plot ofinflammatory marker genes dysregulated in CD14⁺CD16⁻ monocytes from ALSand MS subjects compared to the expression of these genes in CD14⁺CD16⁻monocytes from healthy controls is shown in FIG. 18A (left graph andright graph, respectively). A list of the inflammatory marker genesupregulated or downregulated in CD14⁺CD16⁻ monocytes from ALS and MSsubjects compared to the expression of these genes in CD14⁺CD16⁻monocytes from healthy controls is shown in FIG. 18B.

Example 5. MicroRNAs are also Dysregulated in CD14⁺CD16⁺Monocytes fromALS Subjects

MicroRNA expression profiling was also performed using CD14⁺CD16⁺monocytes from both ALS subjects (n=11) and healthy controls (n=8) (FIG.19). These data show that hsa-miR-708 is increased in CD14⁺CD16⁺monocytes from ALS subjects as compared to the expression of thismicroRNA in CD14⁺CD16⁻ monocytes from healthy subjects.

nCounter expression profiling was performed to identify additionalmicroRNAs dysregulated in CD14⁺CD16⁺ monocytes from ALS (n=8) and MSsubjects (n=8) compared to the expression of microRNAs in CD14⁺CD16⁺monocytes from healthy subjects (n=8). The data in these experimentswere normalized against a geometric mean of five different house-keepinggenes (ACTB, B2M, GAPDH, RPL19, and RPL10). In these experiments, theexpression of 664 microRNAs were analyzed (FIGS. 20A-C). A heatmap ofthe relative expression of microRNAs in CD14⁺CD16⁺ monocytes from ALSsubjects and MS subjects compared to the expression of these microRNAsin CD14⁺CD16⁺ monocytes from healthy controls is shown in FIG. 21A andFIG. 20B, respectively. A summary of the microRNAs significantlyderegulated in CD14⁺CD16⁺ monocytes from ALS and MS subjects compared tothe expression of these microRNAs in CD14⁺CD16⁺ monocytes from healthycontrols is shown in FIG. 20C.

Example 6. Proinflammatory Markers Expressed in Ly6C^(Hi) Monocytes andCD39 ⁺ Microglia from SOD1^(G93A) Mice

The gene expression profile of Ly6C^(Hi) monocytes isolated from thespleen of SOD1 mice one month prior to clinical disease onset and duringdisease progression. Pro-inflammatory genes were expressed at both timepoints (FIG. 21A). Out of 179 inflammatory marker genes measured bynCounter, 97 were detected as having altered expression (compared toLy6C^(Hi) monocytes from non-transgenic litermates): 40 genes wereupregulated in Ly6C^(Hi) monocytes from SOD1 mice (as compared tonon-transgenic litermates) at least one disease stage. Seven genes weredownregulated in Ly6C^(Hi) monocytes in SOD1 mice compared toLy6C^(Hi)monocytes from non-transgenic litermates, including TGFβ1 andthe TGFβ1 receptor (FIG. 21B). Biological network analysis demonstratesthat the most significantly affected pathways in the present analysisrelative to inflammatory responses, including CREB1, NF-kB, PU.1, andPPARy (FIG. 21C). These pathways have been shown to play an importantrole in both monocyte activation and differentiation. The geneexpression profiling demonstrates an activated pro-inflammatoryLy6C^(Hi) monocyte population in the peripheral immune compartment ofSOD1 mice.

Expression profiling of CD11b⁺/CD39⁺ microglia isolated from the spinalcord and brains of SOD1 mice was performed at different stages ofdisease. Out of 179 inflammatory marker genes, 120 were detected: 20genes were upregulated in CD11b⁺/CD39⁺ microglia from SOD1 mice(compared to CD11b⁺/CD39⁺ microglia from non-transgenic litermates)(FIG. 21D) and 38 genes were downregulated in CD11b⁺/CD39⁺ microgliafrom SOD1 mice (compared to CD11b⁺/CD39⁺ microglia from non-transgeniclitermates) (FIG. 21E). CD11b⁺/CD39⁺ microglia microglia from SOD1 miceas compared to the same cells in non-transgenic litermates had prominentexpression of genes related to chemotaxis (e.g., CCL2, CCL3, CCL4, CCL5,CXCR4, and CXCR10). Interestingly, TGFβ1 and the TGFβ1 receptor wereamong the downregulated genes. Biological network analysis demonstratedactivation of inflammatory pathways with the most significant beingchemotaxis (FIG. 21F). The expression of these genes preceded symptomonset and was observed in the spinal cord, but not in the brain (FIG.21G).

Example 7. Proinflammatory Markers Expressed in CD14⁺CD16⁻ Monocytes inALS Subjects

Immune-related gene expression in CD14⁺CD16⁻ monocytes from ALS subjectswas analyzed as described in Example 6. Several inflammatory-relatedgenes were upregulated in CD14⁺CD16⁻ monocytes from ALS subjects ascompared to healthy controls. Although there were some differences inimmune-related gene expression between CD14⁺CD16⁻ monocytes from ALSsubjects and MS subjects, the immune-related gene expression pattern inCD14⁺CD16⁻ monocytes from ALS subjects and MS subjects were similar(FIG. 22A-C).

In a further set of experiments, the expression of 511 immune-relatedgenes was analyzed in CD14⁺CD16⁻ monocytes from subjects having ALS(sporadic and familial ALS). These experiments were performed usingquantitative NanoString nCounter technology. The data gathered fromthese experiments and the data described in Example 6 were furtheranalyzed using GeneGo and Ingenuity® pathway analysis.

The differentially upregulated genes in spinal cord CD39+ microglia andsplenic Ly6C^(Hi) monocytes in SOD1 mice, and in blood-derivedCD14⁺CD16⁻ monocytes from sporadic ALS subjects vs. healthy controlswere analyzed using GeneGo Metacore pathway analysis (GeneGO, St.Joseph, Mich.). This method identifies transcripts that areoverrepresented in defined ontologies. A false discovery rate (FDR)filter was applied to preliminary P values using q-value calculation.After enrichment, the P values were calculated for all the terms withinthe given ontology and each term was tested as a separate hypothesis.The resulting q-values represent corrected P values with an account ofthe total terms in the given ontology and the rank order of theparticular term. The identified significantly dysregulated genes werefurther analyzed to identify biological/disease processes and theinvolved pathway/networks in SOD1 mice and human ALS. The whole data setof 58 dysregulated genes in CD39⁺ microglia from the spinal cord and 47dysregulated genes in Ly6C^(Hi) splenic monocytes from SOD1 mice wereimported into MetaCore to build an analysis of functional ontologiesusing GeneGo process, GeneGo disease process, canonical pathway maps,and networks. The calculation of statistical significance throughoutMetaCore for the maps, networks, and processes were based on P values,which were calculated based on a hypergeometric distribution. P valuesrepresent the probability of a particular mapping arising by chance,given the numbers of genes in the set of all genes present in themaps/networks/processes, the genes on a particular map/network/process,and the genes in the experiment. A P value of 0.01 was used for thecutoff. The degree of relevance for the different categories of theuploaded data sets is defined by P values, so that the lower P valueindicates higher priority. The experimental data were input to build thenetworks. The three different scoring functions that were used to rankthe small subnetworks created by the network building algorithms werezScore, gScore, and p value. The zScore ranks the subnetworks (withinthe analyzed network) with regard to their saturation with genes fromthe experiment. A high zScore means the network is highly saturated withidentified dysregulated genes from the experiment. In other words, itmeans that relatively larger number of genes/analytes in a particularnetwork were present in the aqueous sample. Each network is comprised ofcanonical pathways used to build the network. If a network has a highgScore, it is saturated with expressed genes (from the zScore), and itcontains many canonical pathways. The analysis was controlled formultiple testing by estimating the false discovery rate. Out of 664microRNAs measured, 56 were confidently detected, and twenty weredifferentially expressed in at least one disease group.

Targetscan 14.1 was used to investigate the statistical significance ofmiRNA-mRNA interactions. Targetscan 14.1 was used for the prediction of862044 conserved miRNA binding sites with non-zero context score whichis a measure of conservation. In the SOD1 mouse data set: miRNA targetfiltering analysis using Ingenuity® pathway analysis (IPA) results in 34miRNA families that are predicted to target 10797 mRNAs. These data werefiltered to include only those genes involved in the IPA CanonicalPathway categories representing signaling pathways involved in cellularimmune response, humoral immune response, and cytokine signaling. Thisresulted in filtering of the 34 microRNAs to target 971 mRNAs possiblyinvolved in immune response signaling. The mRNA expression studies wereintegrated using the Nanostring platform into the analysis. 971 filteredtargets contain the 47 immune-related genes that are dysregulated inSOD1 mice taking into account the opposite nature of the miRNA-mRNAregulation. This resulted in a final 87 pairs of miRNA-mRNA interactionsrepresenting 27 miRNA families and 33 mRNAs. In the miRNA expression ofALS subjects study, 56 miRNAs were found to be significantlydysregulated in ALS subjects. Filtering the predicted 862044 sites tothose containing only targets of these 56 miRNAs lead to a reduction inthe number of predicted sites (a reduction to 34118 sites). The numberof sites was further reduced by limiting the data to mRNA targets togenes found to be regulated with a fold change of >1.4 in theimmunological panel nanostring arrays. The final data indicate 68 uniquemiRNA-mRNA interaction pairs in which the mRNA and miRNA are oppositelyregulated. The statistical significance of these 68 miRNA-mRNAinteractions formed by 56 dysregulated miRNAs were further assessed asfollows: 1) 1000 random networks in which 56 randomly selected,non-regulated miRNAs from the study were used to find mRNAs whichcontained a 3′-UTR motif for their binding, and 2) the miRNA-mRNA pairswere further filtered to contain only those 59 mRNAs that weredysregulated in ALS subjects. A mean of 44.88 interactions was observed(SD=9.99). The true interactions determined in the expression studies is68, and corresponds to a significant P value (<1.1×10⁻¹⁵ ). A similaranalysis for the regulated miRNA-mRNA pairs in the SOD1 mice shows aninteraction distribution with a mean of 15.26 (SD=4.03), whereas thetrue miRNA-mRNA interactions determined experimentally is 41, with asignificant P-value of <5.7×10⁻⁹.

The data show that CD14⁺CD16⁻ monocytes from ALS subjects have uniqueexpression of immune-related genes as compared to CD14⁺CD16⁻ monocytesfrom healthy controls. In addition, a few immune-related genes aredifferentially expressed in CD14⁺CD16⁻ monocytes from sporadic ALSsubjects compared to CD14⁺CD16⁻ monocytes from familial ALS subjects(FIGS. 23A-C). These results were validated using singleplex qPCR in anindependent cohort of ALS patients and healthy controls (the changes inthe expression of CCL2, AHR, PTAFR, NF-KB, TRAF3, FCER1A, CXCR4, andSOCS1 were validated) (FIG. 24). These data confirm that CCL2, AHR,PTAFR, NF-KB, and TRAF3 are upregulated in CD14⁺CD16⁻ monocytes from ALSsubjects as compared to CD14⁺CD16⁻ monocytes from healthy controls, andthat CXCR4 and SOCS1 are downregulated in CD14⁺CD16⁻ monocytes from ALSsubjects as compared to CD14⁺CD16⁻ monocytes from healthy controls.

Ingenuity microRNA-mRNA target filter analysis further revealed that thetop 10 microRNA-miRNA interactions in Ly6C^(Hi) cells from SOD1 micewere linked to the genes found to be the most significantly dysregulatedin CD14⁺CD16⁻ monocytes from ALS subjects (FIG. 25).

A further assessment of both the miRNA and mRNA expression profile inCD14⁺CD16⁻ monocytes from ALS subjects shows that the abnormalitiesrelated to miRNA and gene expression in CD14⁺CD16⁻ monocytes is linkedto inflammatory- and immune-related genes (FIG. 26 and FIG. 27). Whenthese miRNA-mRNA interactions in CD14⁺CD16⁻ monocytes were analyzed, theinteractions were shown to be statistically significant using Targetscan4.1 prediction analysis both in SOD1 mice and in ALS subjects (FIG. 28).Furthermore, GeneGo pathway analysis identified 9 inflammation-relatednetworks (FIG. 29). These inflammation networks were identical to thosethat were observed (in the studies described herein) to be dysregulatedin Ly6C^(Hi) monocytes in SOD1 mice.

Example 8. Therapeutic Role of miR-155 in the SOD 1^(G93A) model

Significant upregulation of miR-155 occurs in spleen-derived Ly6C^(Hi)monocytes and spinal cord-derived microglia before clinical onset, whichincreased during all stages of disease progression in SOD1^(G93A) mice(see data above). Additional experiments were performed to determinewhether miR-155 plays a role in the development/pathogenesis of ALS. Inthese experiments, the SOD1 mouse (a model of ALS) was furthergenetically manipulated to knockdown or knockout expression of miR-155.

Animals and Behavioral Analysis

B6/SJL-SOD1^(G93A) Tg and SOD1-wild type (WT) were provided byPrize4Life or purchased from the Jackson Laboratories. ALS mice wereanalyzed at day 30 and 60 (presymptomatic), day 90-100 (earlysymptomatic), and day 120-140 (late symptomatic/end- stage) time points.Onset of symptoms was defined by the peak of the weight curve andvisible signs of muscle weakness. End-stage disease was determined bysymptomatic progression and animal care guidelines (thus it varied fromthe 135 time-point by ±5 days). Disease progression was documentedaccording to established methodology provided by Prize4Life and TheJackson Laboratory. Symptomatic analysis was conducted by dailymonitoring and weight measurements every 3-4 days starting at day 80.Symptomatic onset was defined as the age at which animals began todecline in weight. Neurological scores for both hind legs were assesseddaily for each mouse beginning at 50 days of age. The neurological scoreused a scale of 0 to 4 developed by ALS Therapy Development Institute(ALSTDI). Criteria used to assign each score level were: 0=fullextension of hind legs away from the lateral midline when the mouse issuspended by its tail, and mouse can hold this position for 2 seconds,suspended 2-3 times; 2=collapse or partial collapse of leg extensiontowards lateral midline (weakness) or trembling of hind legs during tailsuspension; 2=curling of the toes and dragging of at least one limbduring walking; 3=rigid paralysis or minimal joint movement, foot notbeing used for forward motion; and 4=mouse cannot right itself within 30seconds from either side, euthanasia.

Generation of SOD1^(G93A)/miR-155^(−/−)

Male SOD1^(G93A)[B6.Cg—Tg(SOD1^(G93A))1Gur/J] mice were bred with non-TgC57B1/6 miR155^(−/−) females. Non-transgenic miR155^(−/−) were backcrossto F1-SOD1^(G93A)/miR155^(−/+) to produce F2-SOD1^(G93A)/miR155^(−/−)with a deletion of miR155. The mice were assessed clinically byneurobehavioral testing (rotarod performance and neurologic score) andthe survival of three experimental groups of SOD1 mice with differentexpression levels of miR-155 was assessed: 1) SOD1^(G93A)/miR155^(+/+);2) SOD1^(G93A)/miR155^(−/+); and 3) SOD1^(G93A)/miR155^(−/−).

Targeting of miR-155 in SOD1 Mice

To prove a direct interaction between miRNAs and their targets, aLuciferase reporter bearing 3′UTR with potential miRNA binding sites isutilized. Site-directed mutagenesis of the miRNA binding site abolishesresponsiveness of the Luciferase reporter to miRNA modulation, whichwill provide proof of direct targeting.

Flow Cytometry

Mononuclear cells were directly isolated from the spinal cord of mice asdescribed in Cardona et al. (Nat. Protoc. 1:1947-1951, 2006) except thatno dispase was used as we found that dispase cleaves several surfacemolecules and can diminish surface detection of surface molecules. Micewere transcardially perfused with ice cold phosphate-buffer saline(PBS), and the spinal cords and brains were separately dissected. Singlecell suspensions were prepared and centrifuged over a 37%/70%discontinuous Percoll gradient (GE Healthcare), mononuclear cells wereisolated from the interface, and the total cell count determined. Thecells were pre-blocked with anti-CD16/CD32 (Fc Block BD Biosciences),and stained on ice for 30 minutes with combinations of anti-Ly6C-FITC,CD11b-PE-CyTM7, and 4D4-APC (unique microglial antibody). 7AAD-PerCP wasused to detect or exclude early apoptotic and dead cells (BDBiosciences). The appropriate antibody IgG isotype controls (BDBiosciences) were used for all stains. Fluorescence-activated cellsorting (FACS) analysis was performed on a LSR machine (BD Biosciences),and the data subsequently analyzed with FlowJo Software (TreeStarSoftware).

Quantitative NanoString nCounter miRNA/Gene Expression Analysis

Nanostring nCounter technology was used to study the expression of up to800 inflammation-related genes. Multiplexed target profiling of 179inflammation-related transcripts which consist of genes differentiallyexpressed during inflammation and immune responses was also performed asdescribed above.

The resulting data show that SOD1^(G93A)/miR155^(−/−) animals have asignificant delay in disease onset and survival compared to SOD1^(G93A)animals. (Tables 22 and 23, and FIGS. 30-34). The body weight of themice was assessed every 3-4 days starting at day 80, the clinicalneurologic score of the mice was assessed daily, and rotarod performancewas assessed 3 times a week. The data show that genetic ablation ofmiR-155 prolonged survival by 51 days (P<0.0001; FIG. 30), extended timeto reach a neurologic score of two by 49 days (P<0.0001; FIG. 31),enhanced rotarod performance (FIG. 32), reduced body weight loss (FIG.33), and delayed early (P=0.0003) and late (P=0.0004) disease onset(FIG. 34).

TABLE 22 Delayed onset and increased survival in SOD1/miR155^(−/−)(Summary of Preliminary Results SOD1.miR155+/+ SOD1/miR155−/− End-stage145 days At 162 days, still breading

TABLE 23 Cumulative Results of Statistical Analysis of SOD1/miR155^(+/−)and SOD1/miR155^(−/−) Mice Kaplan-Meier Survival Fit Median time (days)P value Females SOD1/miR-155^(+/−) SOD1/miR-155^(−/−) Change Log-rankWilcoxon Neurologic onset (Score 2) 138 187 49 <0.0001 <0.0001 Peak bodyweight to death  32  80 48 <0.0001 <0.0001 50% survival 156 207 51<0.0001 <0.0001 Median time (days) P value Males SOD1/miR-155+/−SOD1/miR-155−/− Change Log-rank Wilcoxon Neurologic onset (score 2) 144168 24 <0.0001 0.0003 Peak body weight to death  56  64  8 0.1397 0.064750% survival (age at death) 157 184 27 <0.0001 <0.0001

SOD1^(G93A)/miR155^(−/−) animals also have a significantly reduction inthe recruitment of peripheral monocytes associated with microgliaprotection in the spinal cord, as compared to SOD1^(G93A) mice (FIG.35), and significant reduction in inflammation-related gene expressionin spinal cord microglia and Ly6C^(Hi) monocytes as compared toSOD1^(G93A) mice (FIG. 36). Fewer inflammation-related genes wereaffected in splenic T cells, however, the expression of anti-inflammatory genes (IL4 and IL10) reverted to the level ofnon-transgenic mice, suggesting that miR-155 may primarily affectactivation of the M1-associated signature in Ly6C^(Hi) monocytes inSOD1^(G93A) mice.

These data indicate that miR-155 plays a significant role in thedevelopment (pathogenesis) of ALS, and that treatment of subjects havinga neurodegenerative disorder (e.g., ALS, e.g., familial ALS and/orsporadic ALS) may be achieved by administering at least one aninhibitory nucleic acid targeting hsa-miR-155 (e.g., precursor or maturehsa-miR-155) to a subject a neurodegenerative disorder (e.g., ALS, e.g.,familial ALS and/or sporadic ALS). Exemplary inhibitory nucleic acidstargeting hsa-miR-155 (e.g., precursor or mature hsa-miR- 155) that canbe administered to a subject having a neurodegenerative disorder aredescribed herein.

Example 9. Efficacy of miR-155 antagomir for treating SOD 1^(G93A) mice

A first set of experiments was performed in the SOD1^(G93A) model offamilial ALS to determine whether an antagomir targeting miR-155 wouldalter miRNA expression and/or inflammatory gene expression in spinalcord-derived microglia and splenic Ly6C^(Hi) monocytes. In theseexperiments the following five experimental groups were studied.

-   -   Group I. Control scrambled miR-155 (intraperitoneal injection, 2        mg per injection, every third day) (n=3). Control scrambled        miR-155: +TC+AA+C+A+TTA+G+A+CT+T+A (SEQ ID NO: 263) (“+”        indicates the presence of an LNA moiety).    -   Group II. Antagomir miR-155 low dose (intravenous injection, 0.2        mg per injection, every third day) (n=3). Antogomir miR-155:        +TC+AC+A+A+TTA+G+C+AT+T+A (SEQ ID NO: 262) (the “+” indicates        the presence of an LNA moiety).    -   Group III. Antagomir miR-155 high dose (intravenous injection, 2        mg per injection, every third day) (n=3).    -   Group IV. Antagomir miR-155 low dose (intraperitoneal injection,        0.2 mg per injection, every third day) (n=3).    -   Group V. Antagomir miR-155 high dose (intraperitoneal injection,        2 mg per injection, every third day) (n=3).

Nanostring inflammatory gene and miRNA expression analysis was performedin spinal cord derived microglia and splenic Ly6C′ monocytes asdescribed above.

A comparison of the data from microglia and splenic Ly6C monocytes fromSOD1 mice administered a low (0.2 mg/kg body weight per injection) vs.high dose (2 mg/kg body weight per injection), every third day (by i.p.or i.v.) show that the low dose doesn't affect splenic Ly6C^(Hi)monocyte M1-phenotype (the same miRNA and inflammatory gene expressionis observed in the these mice as compared to untreated SOD1 mice).However, the high dose (administered by either i.p. or i.v.) inhibitedthe expression of pro-inflammatory cytokines as measured by quantitativenCounter technology for inflammation-related genes. The data also showthat spinal-cord derived microglia were not affected by systemicantagomir miR-155 treatment.

A second set of experiments is performed to determine the effect ofantagomir miR-155 on the behavior and survival of SOD1 mice. In theseexperiments, SOD1 mice are either administered a scrambled miR-155(n=10) or an antogomir miR-155 by intraperitoneal injection at 2 mg/kgbody weight per injection, every third day (n=10). The treatment isinitiated at the time of disease onset (defined by body weight loss andneurologic score). The mice are treated continuously until the end ofthe experiment or end-stage. The behavior of the mice is determined, forexample, by rotarod performance, and the body weight and survival of themice is monitored.

A third set of experiments is designed to investigate whether miR-155 inthe central nervous system of SOD1 mice can be targeted usinglentivirus-mediated inhibition of miR-155. In these experiments, theantigomer miR155 is delivered by lentivurus infection. For miR-155inhibition, a sequence encoding mutant miR-155 or its specific inhibitoris cloned in a lentiviral vector (Genecopoeia). The virus is produced byinfecting target cells according to the user's manual. Approximately2×10⁷ transforming units of recombinant lentivirus is delivered to theSOD1 mice by stereotaxic injection to the CSF or the lateral ventricle.The treatment groups are:

Group I. Mice administered a control lentivirus-scrambledmiR-155-GFP-tagged high dose (n=10). (See, control scrambled antagomirsequence of SEQ ID NO: 263.)

Group II. Mice administered an antigomer lentivirus miR-155—GFP taggedhigh dose (n=10). (See, antogomir miR-155 sequence of SEQ ID NO: 262.)

The behavior of the mice is followed, e.g., by rotarod performance, andmonitoring the body weight and survival of the mice. Nanostring miRNAand immune-related gene profiling of the innate immune system and T-cellinflammatory-related gene profiling is also performed on cells derivedfrom these mice (e.g., peripheral Lys6C^(Hi) cells).

nCounter expression analysis was also performed to determine theexpression of several microRNAs in Ly6C^(Hi) spleen-derived monocytesubsets from wild type, SOD1/miR155^(−/+), SOD1/miR155^(−/−) mice. Thedata show that several microRNAs were differently expressed in wild typemice compared to the SOD1/miR155^(−/+) mice, and between theSOD1/miR155^(−/+) mice and the SOD1/miR155^(−/−) mice (FIG. 37).

nCounter expression profiling of blood-derived CD14⁺CD16⁻ monocytes formicroRNAs from sporadic ALS (8 human subjects) and relapsing-remittingmultiple sclerosis (8 human subjects) compared to the expression of themicroRNAs in CD14+CD16− monocytes from healthy controls (8 subjects) wasalso performed. The resulting heatmap in FIG. 38 shows the results ofanalysis of variance (ANOVA) using Dunnett's post hoc test (P<0.01). ThemicroRNAs upregulated or downregulated in CD14⁺CD16⁻ monocytes from ALSsubjects (as compared to expression of these microRNAs in CD14⁺CD16⁻monocytes from healthy controls) are indicated.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims. This listing of claims replaces all prior versions andlistings of claims in the application.

Listing of claims: 1.-39. (canceled)
 40. A method of treatingamyotrophic lateral sclerosis (ALS) in a subject, the method comprisingadministering to the subject an inhibitory nucleic acid comprising asequence that is complementary to a contiguous sequence of at least 7nucleotides present in microRNA hsa-miR-155.
 41. The method of claim 40,wherein the at least one inhibitory nucleic acid is an antagomir. 42.The method of claim 41, wherein the antagomir has a sequence of SEQ IDNO:
 262. 43. The method of claim 40, wherein the at least one inhibitorynucleic acid is an antisense oligonucleotide.
 44. The method of claim40, wherein the at least one inhibitory nucleic acid is a ribozyme. 45.The method of claim 40, wherein the at least one inhibitory nucleic acidis injected into the cerebrospinal fluid of a subject.
 46. The method ofclaim 45, wherein the injection is intracranial injection.
 47. Themethod of claim 45, wherein the injection is intrathecal injection. 48.The method of claim 40, wherein the at least one inhibitory nucleic acidis complexed with one or more cationic polymers and/or cationic lipids.